MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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Unit V: Microbial Water Quality Analysis
Activities:
8.1 Water Testing Sampling Design Discussion
9.1 Fecal Coliforms by Membrane Filtration (Ex 13)
9.2 Most Probable Numbers of Total Coliforms (Ex 14)
9.3 Heterotrophic Plate Count of Water Samples (Ex 15)
9.4 Total Direct Count of Water Samples (Ex 16)
10.1 Coliform Confirmations (Ex 17)
11.1 Water Testing Data Compilation & Discussion
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p 15
p 17
8.1 Water Testing Sampling Design Discussion:
During next weeks lab we will enumerate bacteria in water samples of your choice of origin.
We’ll use different techniques to assess fecal coliform, coliform bacteria, heterotrophic bacteria, and
total bacteria counts. Coliform bacteria are a class of bacterial genera often associated with soil and
fecal contamination. Fecal Coliform bacteria are a subgroup of the Total Coliform bacteria
differentiated by there ability to grow at a higher temperature which mimics that of a mammalian
intestinal tract. We’ll use a membrane filtration technique in determining Fecal Coliform numbers
and the multiple tube technique to estimate the most probable number (MPN) of Total Coliform
bacteria. Numbers of heterotrophic bacteria in the sample will be determined by a modified viable
plate count; whereby, agar pours are used rather than the spread plate technique. Nucleic acid staining
and epifluorescence microscopy will be employed to determine the total bacterial counts in your
samples. You will need to collect your own water sample for these and other experiments prior to
coming to lab next week.
We will co-ordinate sampling within our lab section so to address a particular question. By
today, we will need to discuss a hypothesis, or objective, and design a sampling plan accordingly.
Here are a few suggestions you may consider and build on:
* Storage temperature or time influences on your results for a particular site.
* Assess variation in land use or effluent inputs along a stream.
* Compare two reservoirs used for our drinking water.
* Compare manmade ponds with and without exposure to livestock.
Materials:
Each student will need to pickup supplies for a water sampling kit to take home with you at the
end of class. Place in a ZipLock bag, a pair of latex gloves of your size and 2 WhirPak bags, which
you must label with your initials. Keep gloves and WhirPak bags sealed in the ZipLock until you are
ready to sample.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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Procedure:
Collect your water as outlined below and demonstrated by your instructor. Collect no sooner
than 24 h prior to the lab. The fresher the sample the better; however; your sample is part of a class
effort, so it is important to coordinate a common sample time and sample storage condition as
these factors can bias results. Wear latex gloves when sampling to protect yourself (e.g., if you
were sampling toilet water) and/or your sample from outside contamination from your hands (e.g., if
you sample natural waters or tap water low in bacteria). Take two samples from the same site (one is
a back up) following the instructions below:
1) Wear gloves.
2) Rip off the perforated seal on a WhirPak bag. The bag is sterile inside.
3) Holding the bag via the white tabs, lower it below the water surface or hold it under the flow
of water, depending on your site choice.
4) Open the bag by pulling out on the white tabs. Pull the bag through the water to fill the bag,
or let water flow into the bag, until the bag is about 2/3 full.
5) Holding the bag by the yellow twist ties, whirl your sample over itself about 3 times (see
demonstration).
6) Fold the ends of the yellow twist ties together and twist a few times.
7) Repeat steps 2-6 for your second sample, which we’ll collect as a backup sample.
8) Store samples upright inside the sealed ziplock bag, and maintain your sample at the same
temperature as your site until lab (avoid excessive warming or cooling of your sample).
9.1 Fecal Coliforms by Membrane Filtration: (Ex 13)
Water that contains large numbers of bacteria may be perfectly safe to drink. The important
consideration, from a microbiological standpoint, is the kinds of microoganisms that are present.
Water from streams and lakes, which contain multitudes of autotrophs and saprophytic heterotrophs,
is potable as long as pathogens for humans are lacking. The intestinal pathogens such as those that
cause typhoid fever, cholera, and bacillary dysentery are of prime concern. The fact that human fecal
material is carried away by water in sewage systems that often empty into rivers and lakes presents a
colossal sanitary problem; thus, constant testing of municipal water supplies for the presence of fecal
microorganisms is essential for the maintenance of water purity.
Routine examination of water for the presence of intestinal pathogens would be a tedious and
difficult, if not impossible, task. It is much easier to demonstrate the presence of some mostly
nonpathogenic intestinal types such as Escherichia coli or Streptococcous faecalis. Because these
organisms are always found in the intestines, and normally are not present in soil and water, it can be
assumed that their presence in water indicates that fecal material has contaminated the water supply.
E. coli and S. faecalis are classified as good sewage indicators. The characteristics that make
them good indicators of fecal contamination are 1) they are not normally present in water or soil, 2)
they are relatively easy to identify, and 3) they survive a little longer in water than enteric pathogens.
If they were hardy organisms, surviving a long time in water, they would make any water purity test
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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too sensitive. Since both organisms are non-spore formers, their survival in water is not extensive, at
least for temperate latitude surface samples.
E. coli and S. faecalis are completely different organisms. E. coli is a gram- negative, nonspore forming rod. S. faecalis is a gram-positive coccus. The former is classified as a coliform, and
the later is an enterococcous. Physiologically, they are also completely different.
By definition, a coliform is a facultative anaerobe that ferments lactose to produce gas when
grown at 37C and is a gram-negative, non-spore forming bacilli (rods). Escherichia coli fits this
description, yet so do many other bacteria commonly found in soils, such as Enterobacter aerogenes
and species of the genera Klebsiella or Citrobacter. S. faecalis is not a coliform, and a completely
different set of tests must be used for its detection. In Exercise 14, we will estimate the most
probable number (MPN) of total coliform bacteria in our sample. Because that value may include
coliform bacteria of non-fecal source we will also assay for a more specific subset of coliforms, the
fecal coliforms. Fecal coliforms are those coliforms that can grow at an elevated temperature of
44.5ºC, and their density in a sample should be lower than total coliforms. Of the genera discussed
above, only E. coli is a fecal coliform.
Our estimate of fecal coliform bacteria will be based on a modified viable count procedure,
called the membrane filtration technique. Because the numbers of fecal coliform bacteria in the
sample are likely to be very low, we need a means of harvesting bacteria from a large volume of water
(100 ml). This volume is well is obviously in excess of what can be spread on an agar plate. To solve
this problem, bacteria in a known volume of water sample are harvested onto a filter membrane by
vacuum filtration. The filter with bacteria is placed, sample-side-up, onto agar media or an absorbent
pad saturated with broth media (Figure 5.1). The media for fecal coliform bacteria, mFC agar, is
selective and differential. Media ingredients easily diffuse through the membrane filter to the
bacteria. Fecal coliform bacteria on the filter will grow at 44.5 ºC and form distinctly blue colonies.
The blue CFUs on the filter divided by the volume of sample filtered will yield an estimate of fecal
coliform bacterial density in the sample.
EXERCISE 13:
Membrane Filtration Technique:
Materials:
Sterile filtration apparatus for 47 mm diameter filters
3 x 47 mm diameter gridded nitrocellulose filters (0.45 µm pore)
forceps
3 x mFC agar media in 50 mm diameter Petri plates
300 mL Sterile distilled water (optional)
Procedure:
We will work in pairs, one pair at a time throughout the lab period, as there is only one filtration
manifold for us to use.
1. Always use sterile technique, including the sterilization of forceps with ethanol, as
demonstrated.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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2. Disassemble the sterile filtration apparatus, and make sure not to contaminate its interior.
3. Place a gridded membrane filter onto each of the three filtration frits of the apparatus and
screw on the towers. Careful not to strip the plastic screw threads.
4. Mix the sample well.
5. Add sterile water using the sterile graduate cylinder and then pipet your known sample to a
total volume of 100 ml as follows:
NOTE: Always add sterile water to the tower first, prior to adding the desired sample volume; use
the graduate cylinder for your 100 ml sample last.
99 ml sterile water, then pipet 1 ml sample, stir with the pipet.
90 ml sterile water, then pipet 1 ml sample, stir with the pipet.
100 ml sample, stir with the pipet; NO sterile used here.
mFC agar
Figure 5.1. Removal of membrane filter from filtration manifold and placement on mFC agar.
(Johnson and Case, 2001; Fig 53.1)
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
6. Apply vacuum until the filter sucks dry. Immediately disassemble the tower from the
apparatus.
7. Using sterile forceps, place the membrane filters, sample-side-up, onto mFC agar of the 50
mm diameter Petri dishes (Figure 5.1). Avoid trapping air bubbles between filter and agar.
8. Incubate at 44.5ºC for 24h to 48 hours.
9. Count colonies on plates with 1 to 100 colonies, and calculate sample density by dividing by
sample volume for each filter. Take an average of results if more than one plate is in the 30300 CFU range.
10. Share the date with the class as directed by the instructor. Address all data presentation and
discussion points given under section 11.1 Water Testing Data Compilation & Discussion
on page 17 of this unit.
9.2 Most Probable Numbers of Total Coliforms: (Ex 14)
The presumptive test is used to determine if gas-producing lactose fermenters, coliform
bacteria, are present in a water sample. If, after incubation, gas is seen in any of the lactose broths, it
is presumed that coliform bacteria are present in the water sample (Figure 2). For clear surface water
samples, nine tubes of lactose broth will be used and inoculated with three different volumes of
sample. For turbid surface water, the sample will first need to be diluted 1/10 (see Ex 15 for this
dilution), and this diluted sample is used for inoculation of tubes. In addition to determining the
presence or absence of coliform bacteria, we can also use this series of lactose broth tubes to
determine the most probable number (MPN) of coliform bacteria present in the 100 ml of water. A
table for determining this value from the number of positive lactose tubes is provided (Table 5.1).
EXERCISE 14:
MPN Assay:
Before setting up your test, determine whether or not your water sample is clear or turbid.
This decision will also determine which dilution protocol is used for the heterotrophic plate count
(HPC) in Exercise 15. Note that a separate set of instructions is provided for each type of water.
Materials:
3 Durham tubes of DSLB
6 Durham tubes of SSLB
1 10 ml pipette
1 1 ml pipette
Note: DSLB designates double strength lactose broth. It contains twice as much lactose as SSLB
(single strength lactose broth).
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MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
Determine if any sample dilution is required:
If you sample is turbid (cloudy) you will need to use the 1/10 dilution of your sample from Ex 15 on
heterotrophic plate counts for inoculating your MPN tubes. If your sample looks clear, then use your
sample directly as inoculum for your MPN tubes. Ask the instructor if you are uncertain.
Figure 5.2. Presumptive phase in determining the most probable numbers (MPN) of coliform
bacteria in a water sample (modified from Benson, 1994; Fig 61.1).
Procedure:
1) Set up three DSLB and six SSLB tubes as illustrated in Figure 5.2. Label each tube according to
the volume of water used to inoculate it: 10 ml, 1.0 ml, and 0.1 ml, respectively.
2) Mix the sample (clear water sample) or 1/10 diluted sample (turbid water sample) well.
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MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
3) With a 10 ml pipette, transfer 10 ml of inoculum to each of the DSLB tubes.
4) With a 1.0 ml pipette, transfer 1 ml of inoculum to each of the middle set of tubes.
5) With a 1.0 ml pipette, transfer 0.1 ml of inoculum to each of the last three SSLB tubes.
6) Incubate the tube at 35 oC for 24 hours.
Table 5.1. Most probable number (MPN) determination for a 3–replicate tube test for total coliform
bacteria (Bensen, 1994).
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MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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7) Examine the tubes and record the number tubes in each set that have 10 % gas or more.
8) Determine the MPN by referring to Table 8.1. If you used the 1/10 diluted sample as the MPN
inoculum because your sample was turbid, then your result from the MPN table needs to be multiplied
by 10 to account for the dilution. This will give you the result for the original sample.
EXAMPLE: Assume you inoculated tubes with the original sample. After incubation, you see gas in
the first (“10 ml”) three tubes, gas is only in one tube of the second series of tubes (“1 ml”), but no
gass I seen in any of the last three tubes (“0.1 ml”). Your test would read as 3-1-0. Finding this
combination in Table 8.1 gives an MPN of 43 CFU per 100 ml, and a 95% probability of there being
between 7-210 CFUs per 100 ml. Keep in mind that an MPN value is only a statistical probability.
9) Share the date with the class as directed by the instructor.
10) Address all data presentation and discussion points given under section 11.1 Water Testing
Data Compilation & Discussion on page 17 of this unit.
9.3 Heterotrophic Plate Count of Water Samples: (Ex 15)
Determining the total numbers of viable bacteria in water is very difficult. Aquatic bacteria
have great variability in physiological needs, and no single medium, redox potential, pH, or
temperature is ideal for all types. Keep in mind, that the same problems are encountered with
enumerating bacteria in soil. In spite of the fact that only small numbers of heterotrophic bacteria in
water will grow on non-selective nutrient media, the Heterotrophic Plate Count (HPC) can perform an
important function in water testing. Probably its most important use is to give us a tool to reveal the
effectiveness of various stages in the purification of water or to identify eutrophic conditions in
natural aquatic ecosystems. For example, HPCs made of water before and after storage can tell us
how effective holding is in reducing bacterial numbers. Likewise, HPCs of river water upstream and
downstream from a potential source of mineral nutrient or organic pollution can reflect the magnitude
of ecosystem disturbance. In this exercise, your samples of water will be evaluated by a routine HPC
procedure. Because different dilution procedures are required for different types of water, you will
need to inform the instructor of your sample source so they can direct you to follow either the low or
high dilution protocol (Figure 5.3) below.
EXERCISE 15:
Heterotrophic Plate Count:
Materials:
1.0 ml pipettes
10 ml pipettes
beaker for holding your Whirlpak with sample
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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3 bottles of sterile water for dilutions
6 tubes with 15 ml liquefied (45C) tryptone glucose extract agar (TGEA) pours
6 sterile Petri plates
Quebec colony counter and hand counters
water samples collected as instructed last week
Procedure:
1. Your 6 tubes of liquefied TGEA are in the 45 ° C water bath. Keep them there until step 6.
2. Determine whether you will use the “high” or “low” dilution protocol and label the sterile Petri
dishes with your initials and final dilution factor (i.e. volume of original sample plated). You should
have 2 replicate plates for each of three final dilution factors (Figure. 5.3).
3. Shake the sample of water 25 times.
4. Make necessary dilution as indicated in the protocol you require. A 10 ml volume will be
transferred into a bottle of 90 ml sterile water and mixed well to make 10-fold serial dilutions:
High Dilution Protocol required 3 serial transfers to use the 10-3, 10-2, and 10-1 DF.
Low Dilution Protocol required 2 serial transfers to use the 10-2, 10-1 and 1 DF (sample).
5. For each dilution (or sample) used, pipette 1 ml to each of the two sterile Petri plates.
6. Using one tube of liquified TGEA per plate, pour the medium into the dishes, rotate and rock
sufficiently to get good mixing of medium and diluted sample, and let cool to solidify.
7. Incubate at 35 ° C for 24 hours.
8. Select the pair of plates that has 30 to 300 colonies on each plate and count all the colonies on both
plates. Record the average count for the two plates and calculate the density of heterotrophic bacteria
(= CFU/final DF in ml) in your original water sample.
9. Share the date with the class as directed by the instructor.
10. Address all data presentation and discussion points given under section 11.1 Water Testing Data
Compilation & Discussion on page 17 of this unit.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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High Dilution Protocol: (e.g. turbid eutrophic surface water)
10 ml
10 ml
10 ml
90
ml
90
ml
90
ml
10-1
10-2
10-3
Sample
1 ml
10-1 ml
1 ml
1 ml
10-2 ml
10-3 ml
Low Dilution Protocol: (e.g. tap water; clear oligotrophic surface waters)
10 ml
Sam
ple 1 ml
1 ml
10 ml
90
ml
90
ml
10-1
10-2
1 ml
10-1 ml
1 ml
10-2 ml
Figure 3. Dilution series protocols for Heterotrophic Plate Counts.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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9.4 Total Direct Count of Water Samples: (Ex 16)
Bacteria represent a large fraction (5 to 50%) of living biomass in both aquatic and terrestrial
ecosystems. It is the large biomass, activity and metabolic diversity of bacteria compared with other
organisms that make them the driving force in the cycling of elements, such as C and N, in
ecosystems and the biosphere as a whole. An understanding of bacterial biomass is not only
important from the perspective of elemental cycling, but also for assessing bacteria as a source of
energy for growth of organisms at higher trophic levels, like protists or earthworms. We can
determine the total bacterial biomass in a sample by direct observation of bacterial abundance (i.e.
density) and biovolume (i.e. total cell volume) in soil or aquatic samples. However, cell size
measurements used to estimate average cell biovolume and the subsequent algorithms used to
calculate biomass are beyond the scope of today’s lab period. Today we will take some time to
demonstrate the technique and calculations for estimating the abundance of bacteria, i.e. the “total
direct count” (TDC), in water samples.
Estimating TDC in water involves the following: 1) staining the cells in the sample; 2) filtering
a known volume onto a known surface area; 3) mounting the filter with sample on a glass microscope
slide; 4) counting the average number of cells in the microscope field of view; and 5) calculating
abundance using the microscope count, known surface areas (i.e. filtration surface and microscope
field areas) and volume sample (see below).
Conventional basic stains (e.g., crystal violet) and simple light microscopy don’t work well for
visualizing bacteria in environmental samples because cell sizes in nature are variable and can be
smaller (<10%) than that of cultured bacteria. Also, conventional basic stains will not distinguish
between cells and the many amorphous and mineral particles in water or soil samples. Therefore,
fluorescent stains specific for nucleic acids (e.g. acridine orange) are most frequently used to
enhance the sensitivity of detecting all bacteria in these samples. A microscope equipped with a
fluorescent (UV) light source is required to excite fluorescent stains so that we can visualize the
emitted wavelength of visible light and see the cell. Different fluorogenic compounds need certain
wavelengths of UV light to become excited, and the wavelength of visible light (hence color) emitted
is also compound specific. Therefore, the fluorescence microscope must have the appropriate set of
exciter and emitter (also called “barrier”) optical filters in order to function when a given fluorogenic
compound is used. An epifluorescence microscope is needed when working with opaque sample
preparations (e.g., filters or soils smears) as proper function does not require the UV light source to
pass through the sample, as is the case for direct-fluorescent microscopy (Figure 5.4).
Epifluorescence microscopes have a UV light source that illuminates the sample surface
(hence “epi-“) from above, which negates the issue of sample transparency.
We will demonstrate preparation of your aquatic samples onto slides and count cells for slides
placed on the epifluorescent microscopes. Below are brief descriptions of the protocols used to
prepare aquatic samples for determining a TDC; a similar protocol is available for TDC of soil
samples. It will be most memorable if you actually attempt to count and calculate the abundance of
bacteria in these samples.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
Epifluorescence
Hg lamp
UV light
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Direct-Fluorescence
ex.
em.
em.
transparent
sample
ex.
opaque
sample
Hg lamp
UV light
Figure 4. Cartoons illustrating light paths for epifluorescence and direct-fluorescence microscopes.
Both exiter filter (ex.) and emission filter (em.) are indicated. Note the ability of the epifluorescence
microscope to function with opaque samples.
EXERCISE 16:
Total Direct Count:
Materials:
20 ml vial
37% buffered formaldehyde (formalin)
0.2% acridine orange solution
pipetters and tips
filtration apparatus for 25 mm diameter membrane filters
25mm diameter filters: black polycarbonate (0.22 µm pore) and nitrocellulose (0.45 µm pore)
forcepts
glass slides and cover slips
immersion oil
epifluorescent microscope
hand tally counter
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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Procedures:
Sample Preservation and Slide Preparation
1) Bacteria in aquatic sample are preserved by simply adding 1 ml of 37% formaldehyde to a 20 ml
sample. These samples can be stored for 2 weeks in a refrigerator.
2) Wet both types of membrane filters (nitrocellulose and polycarbonate) on respective petri dishes
with sterile filtered water.
3) Set-up the filtration apparatus (Figure 5.5). With the tower off, place a nitrocellulose membrane
filter (0.45 m pore size) centered on the base; this will help distribute the vacuum evenly. Next,
place a polycarbonate membrane filter (0.22 m pore size) evenly on top. The polycarbonate
membrane is stained grey/black to reduce background fluorescence during counting. Replace the
tower; gently locking it in place, i.e. only finger tighten. Vacuum valve should be in the off
(horizontal) position.
4) Pipette 2 to 10 ml volume of sample into the bottom of the tower. Record this volume as it is
needed for calculations. Add 1/10th sample volume of 0.2% Acridine Orange stain solution (e.g. a 2
ml sample will require adding 0.2 ml of 0.2% acridine orange). Swirl the filtration apparatus to mix
sample and stain. Incubate for 10 minutes in the dark.
tower
Cover slip
polycarbonate filter
nitrocellulose filter
base
C
oil drop
B
filter
valve
A
(ON position)
slide
to vacuum
Figure 5.5. Filtration apparatus and
membrane filter set-up for total direct counts
of water.
Figure 5.6. Mounting of the polycarbonate
filter to the glass slide with immersion oil.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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The sample with stain is filtered (i.e., the cells are harvested onto the filter) and rinsed by filtration
with about 2 ml sterile filtered water. Remove the moist filter under reduced vacuum and place it on a
glass slide, sample-side-up. Add a drop of immersion oil to a glass cover slip, invert it, and place it oilside-down on the filter (Figure 6). Slides should be counted immediately, or stored frozen in darkness.
Counting using Epifluorescence Microscopy
Before actually counting the bacteria on the slide, we need to measure and calculate the
microscope field of view area (G) and the filtration area on the filter (F). From these areas we can
compute the number of microscope fields it takes to cover the filtration area (= F/G); this value is
needed in our final calculation of TDC. The following steps walk you through these area
measurements, and then proceed to describe how to count and compute TDCs. Steps #1 and #2 below
have been done for you.
1) Using a stage micrometer observed with the 100X oil objective, determine the apparent size of the
net micrometer grid installed in one of the oculars. Calculate the area of this “field of view” [G = grid
width squared = (100 m)2 = 10,000 m2].
2) Measure the inside diameter of the filtration tower bottom, which is slightly smaller than for the
filter itself. Use the value to calculate the filtration area on the filter (F). In our case, the inside tower
diameter is 23 mm, i.e. it has a radius of 11,500 m [F = r2 = (11,500 m)2 = 4.15 x 108 m2].
3) Count the total number of bacteria glowing orange or green in a single field of view using the 100X
oil objective. Remember, the bacteria are very small - usually only several µm in length or diameter!!
You should count between 40 and 60 cells per field of view. If there are more cells per field, then just
count a constant fraction of the grid (i.e. same number of grid rows) and correct for that fraction in
your final calculation by division (Figure 5.7). Count 10 randomly selected fields separately and then
compute an average number of cells per field (N).
4) To determine the Total Direct Counts (TDC) of bacteria per ml of water use the following formula:
TDC (bacteria ml-1) = (F/G)NS-1 = (4.15 x 104 fields)NS-1
G = area of the field of view (i.e. the net micrometer grid)
F = filtration area
N = average number of bacteria counted per field of view
S = volume (ml) of sample filtered
Notice the value for filter area divided by grid (F/G) is given as 4.15 x 104 fields. What this means is
we can fit 41,500 grid areas, i.e. “fields”, seen through the microscope over the surface of the filter’s
filtration area. Multiplying our average count per field by this value gives the total number of bacteria
in our volume of sample filtered. Dividing the later value by the sample volume filter (S) gives the
total direct count. The final TDC value should be about 106 cells/mL, or 109 cells/L.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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TDC example for 2 ml water filtered:
More than 60 bacteria per field of view; counting 3
rows will yield 40-60 bacteria.
100 m
Top 3 rows have 52 bacteria. Correcting for the
field fraction counted gives 173 bacteria per field
[= (10 rows/field) x (52 bacteria/3 rows)].
TDC = (4.15x104 fields)(173 bacteria/field)(2ml)-1
TDC = 3.6 x 106 bacteria ml-1
100 m
Figure 5.7. A view through the epifluorescence microscope using the 100x oil objective.
Note the 10 x 10 row grid, or “field of view”. The sample calculation is based on
counting only the top 3 rows.
5) Share the date with the class as directed by the instructor.
6) Address all data presentation and discussion points given under section 11.1 Water Testing
Data Compilation & Discussion on page 17 of this unit.
10.1 Coliform Confirmations: (Ex 17)
Presumptive testing for the MPN of total coliform bacteria (Exercise 17) requires further use
of selective and differential agar media to confirm that “positive” growth in tubes are in fact coliform
bacteria. Sometimes two or more species of non-coliform bacteria can act together to catabolize
lactose and produce gas; a interaction called synergism. For this reason we will test “questionably
positive” tubes in addition to representative positive results. The assay is said to be completed when
the confirmed bacteria are again tested as coliforms by independent assays.
Confirmation testing involves inoculating plates of Levine EMB agar or Endo agar from
positive (gas producing) tubes to see if the organisms that are producing gas are gram-negative
(another coliform characteristic). Both of these media inhibit the growth of gram-positive bacteria and
allow colonies of coliform bacteria to be distinguishable from non-coliforms. On EMB agar coliforms
produce colonies with dark centers (nucleated colonies). On Endo agar coliforms produce reddish
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
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colonies that may have a metallic green sheen. The presence of coliform like colonies confirms the
presence of a lactose-fermenting gram-negative bacterium.
In the completed test our concern is to determine if the isolate from the agar plates in the
confirmation test truly matches our definition of a coliform. Media for this test includes a nutrient agar
slant and a Durham tube of lactose broth. If gas is produced in the lactose tube and a slide from the
agar slant reveals that we have a gram-negative, non-spore forming rod, we can be certain that we have
a coliform.
The completion of these three tests with positive results establishes that coliforms are present;
however, there is no certainty that E.coli is the coliform present. The organisms might be E.
aerogenes. Of the two, E. coli is the better sewage indicator since E. aerogenes can be of non-sewage
origin. To differentiate these two species, one must perform further tests to identify members of the
Enterobacteraciae, which includes many common and pathogenic intestinal bacteria; we’ll perform
these tests in Unit IX later this semester.
EXERCISE 17:
Confirmation Assays:
Materials:
ENDO agar plates
Levine EMB agar plates
Positive and questionable MPN tubes
Procedure:
You will need to test a representative strong positive tube and any questionably positive tubes from
your MPN assay. Each sample will be plated on both media by streaking for isolation.
1. Label the tubes you use and have the label code coincide with labels you place on the
respective ENDO and EMB plates.
2. Using an inoculation loop and sterile technique, streak each sample for isolation of colonies on
each media type.
3. Incubate EMB and ENDO plates at 37ºC for 24 to 48 h.
4. After incubation check each sample for positive coliform colonies. Record results for each
sample on each media type.
5. Share the date with the class as directed by the instructor.
6. Address all data presentation and discussion points given under section 11.1 Water Testing
Data Compilation & Discussion on page 17 of this unit.
MICRB 202: Introductory Microbiology Lab
Unit V: Microbial Water Quality Analysis
17
11.1 Water Testing Data Compilation & Discussion:
How to exactly proceed with data analysis, presentation and discussion will largely depend on
the objective of your water quality study and sampling design. Below are some general
expectations for data presentation and discussion points to include along with your study specific
discussion.
1) You will work with the pooled class data set for all parameters collected: FC, TC-MPN, HPC,
and TDC.
2) Convert all parameter values to reflect a standard unit; e.g. cells per 100 mL.
3) Average any replicate samples and present means and standard deviations.
4) If you wish to discuss “significant” differences, I expect you to present the statistic and critical
values.
5) Present your data in a graphic form using Excel or SigmaPlot.
6) I expect proper figure legends written beneath your graphs (we’ll discuss this in lab).
7) Was there a decrease in magnitude from TDC to HPC to MPN to FC? Discuss why you
expected this outcome.
8) Discuss the results within the context of your study objective / hypothesis and validity of your
data. Discuss the unexpected.
9) Draw some conclusions relevant to the objective.
10) What might you perform differently next time to yield more rigorous results or if you could
take many more samples.