LAB 4 a
BACTERIOLOGICAL EXAMINATION OF WATER
The quality of potable (drinking) water available for public use is of major concern to
everyone. In densely populated areas, pollution by sewage is an ever-present hazard.
Several serious diseases can be traced to polluted drinking water, among them typhoid
fever, cholera and others in a group of intestinal disorders generally called "dysentery".
It is generally difficult and time consuming to isolate and identify the relevant
human pathogens because of their low numbers. Instead, bacteriological tests of
drinking water establish the degree of fecal contamination of a water sample by
demonstrating the presence of indicator organisms. The ideal indicator organism
should (1) be present only whenever the pathogens concerned are present, (2) be
present only when there is a real danger of pathogens being present, (3) occur in
greater numbers than the pathogens to provide a safety margin, (4) survive in the
environment as long as the potential pathogens, (5) be easy to detect regardless of
what other organisms are present in the sample, and (6) should not multiply in water to
give higher estimates of cell numbers. The bacterial organism that probably satisfies
these criteria best is Escherichia coli. It is a member of the coliform group of bacteria.
Many methods have been developed to detect these indicator organisms
including the most probable number (MPN) technique, a statistical method. However
the most widely used method today is the membrane filter technique. In the membrane
filter technique, the water sample is filtered with the aid of suction through a sterile
membrane with a pore size of 0.45 μm. Bacteria in the sample are trapped on the
surface of the filter. The filter is then carefully removed and placed on an agar surface.
Nutrients pass through the filter, and the cells on the surface develop into colonies large
enough to be counted after 24 or 48 hours incubation. One assumes that each
bacterium trapped on the filter will then grow into a separate colony. By counting the
colonies one can directly determine the number of bacteria in the water sample that was
filtered. The method has the advantage of being able to process large volumes of water
which is especially useful when bacterial counts are low. A disadvantage involves the
processing of turbid specimens; particulate matter clogs the pores and inhibits passage
of the specific volume of water.
The coliform group is aerobic or facultatively anaerobic gram-negative nonspore-forming rods which ferment lactose to produce acid and gas at 37C within 48
hours. The demonstration of the presence of these organisms is aided by the use of
media such as Endo and FC agar. In addition to promoting growth of coliform
organisms, Endo medium is also selective in discouraging the growth of most other
species of bacteria. This is a significant help in these tests because "raw" water
sources contain hundreds of different species of microorganisms having nothing to do
with fecal or sewage pollution. These organisms would otherwise completely overgrow
the test filter and mask the presence of any coliform colonies. When coliform bacteria
break down lactose they form a number of simpler substances, among which are a
group of chemicals known as aldehydes. Endo medium contains lactose and other
nutrients, and also a stain (basic fuchsin) that reacts with the aldehyde molecules and
forms a complex that appears as a shiny green coating. Because few microorganisms
make aldehydes out of lactose, the "green sheen" colonies are quickly identified as
coliform bacteria.
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Three media and two temperatures will be used for the detection of bacteria in
water. The Endo media as described above will be incubated for 24 hours at 37C.
The coliforms show up as colonies with a dull to bright, metallic, green-gold sheen.
Fecal coliforms are identified on FC agar incubated at 44.5C for 24 hours. FC agar
has bile salts for the inhibition of most gram-positive organisms and Rosolic Acid for an
indicator. The colonies that are blue or blue-green are considered to be fecal coliforms.
Fecal streptococci are identified on KF agar incubated at 37C for 48 hours. KF agar
contains sodium azide that inhibits most gram-negative organisms and
triphenyltetrazolium chloride (TTC) as an indicator. TTC causes the enterococci to form
a red color as a result of tetrazolium reduction to an acid azo dye. Red, pink or maroon
colonies are counted as being fecal streptococci.
Fecal streptococci are more resistant to halogen disinfection than are the
coliform organisms. Demonstration of their absence is therefore an indication of
adequate disinfection.
The total number of coliforms allowed in drinking water is 0-5 per 100 ml. Fecal
coliforms and streptococci should be absent from drinking water samples.
Open bodies of water are subject to animal excretion as well as seepage from
soil. The ratio of the two groups in these samples is an indication of the type of
pollution. Fecal coliform (FC) to fecal streptococci (FS) ratios of less than 0.7 suggest
animal fecal contamination and ratios greater than 4.0 may indicate human fecal
pollution. These ratios can only be used when both indicators suggest polluted
conditions, ie. FC greater than 100/100 ml and FS greater than 20/100 ml.
References:
1) Prescott’s Microbiology, 8th Edition, “Water Purification and Sanitary Analysis”
pp.1052-1057
2) Guidelines for Canadian Drinking Water Quality Summary Table. 2010. Health
Canada. (http://www.hc-sc.gc.ca/ewh-semt/alt_formats/hecs-sesc/pdf/pubs/watereau/2010-sum_guide-res_recom/sum_guide-res_recom-eng.pdf)
3) Guidelines for Canadian Drinking Water Quality: Guideline Technical Document:
Total Coliforms. 2006. Health Canada, Ottawa. (Membrane filtration technique on p.
14)
(http://www.hc-sc.gc.ca/ewh-semt/alt_formats/hecs-sesc/pdf/pubs/watereau/coliforms-coliformes/coliforms-coliformes-eng.pdf)
Materials: per assigned group (4)
1.
2.
3.
4.
5.
6.
7.
8.
Water samples for examination.
Filtration apparatus, sterile.
Vacuum pump.
Sterile membrane filters, forceps, dish of alcohol.
Endo, FC and KF media.
Dispensing flask with sterile water.
Sterile 10 ml pipettes.
Sterile 100 ml measuring cylinder.
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Method: DAY 1
1.
The size of the sample filtered depends on the expected number of coliforms.
Rough guides will be given below.
2.
Assemble the filter apparatus aseptically as follows: place the filter holder
securely in the receiving flask, then using flamed forceps transfer a filter to the
porous glass support, place the funnel holder on top and secure with the clamp
provided. Connect the filter flask to the vacuum pump.
3.
Filter 50 ml of sterile water, remove the clamp and funnel holder carefully,
remove the filter using flamed forceps and place on Endo media. This is your
control.
4.
Test specimens:
(a)
Agitate the sample well.
(b)
Aseptically place another sterile membrane into the filter assembly.
(c)
Filter the required volume of water. For small volumes be sure to add
sterile water to the funnel as you pour or pipette in the sample so as to
increase the fluid volume (this will be demonstrated).
(d)
Aseptically remove the filter and place with a rolling motion onto the
surface of the specified plate.
(e)
Be sure to rinse the apparatus with sterile water after completing each
sample. Also be sure to filter the smaller volume of the two first when it is
necessary to filter two aliquots of different volumes.
Samples:
(a)
Kingston tap water
Filter 100 ml, plate on TSBA
Filter 100 ml, plate on Endo.
(b)
Well water
Filter 100 ml, plate on TSBA
Filter 100 ml, plate on Endo
(c)
Lake Ontario water
Filter 10 ml, 1 ml, 0.1 ml; Plate on TSBA.
Filter 100 ml, 10 ml, 1 ml, 0.1 ml; Plate on Endo.
Filter 100 ml and 10 ml; Plate on FC.
Filter 100 ml and 10 ml; Plate on KF.
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DAY 2.
5.
Count the plates after the prescribed incubation times. (The TA will arrange a
common time for all the students to return to count your plates.) Record
background colony counts on each plate as well as coliforms, fecal coliforms and
streptococci. Determine the FC:FS ratio if possible.
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Student #: __________________
Questions (Hand in)
1.
List all data (see Day 2, step 5) in tabular form (example shown below). Express
calculated data as # per 100 mL.
Sample
2.
dilution
No of colonies
on plate
CFU/100 mL of
sample
Is your data accurate? If not, comment on the source(s) of error.
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3.
Use the information provided on the Health Canada website (http://www.hcsc.gc.ca/ewh-semt/pubs/water-eau/2010-sum_guide-res_recom/index-eng.php) and
your own results to complete the table below. Comment on the potability of all the water
samples based on the Health Canada microbiological guidelines.
Microbiological
parameter
Health Canada
Guidelines
Maximum
acceptable
concentration
(CFU / 100 mL)
Heterotrophic
plate count
Total coliform
Fecal coliform (E.
coli)
Fecal streptococci
Giardia and
Cryptosporidium
Enteric viruses
Measured values (CFU/100 mL)
Kingston tap
water
Lake Ontario
water
Well water
(indicate
sample done)
Not determined
Not determined
Not determined
Not determined
Not determined
Not determined
Not applicable
Comment on
potability
4.
In most rural homes that obtain drinking water from a well, the water is pumped
from the well to the house. If a resident’s well water has a low level of fecal
coliforms present, recommend how the water may be treated to make it potable.
Justify your choice.
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5.
In certain communities, the presence of a protozoa, Giardia or Cryptosporidium,
is a potential problem.
(i) What disease do they cause?
(ii) What technology is commonly used to remove these organisms from drinking
water. Explain the underlying principle by which it works.
6.
What are disinfection-byproducts and what is their impact on human health?
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LAB 4 b
HISTOLOGICAL EXAMINATION OF CELLS, TISSUES, AND
ORGANS
Clinicians and researchers commonly examine prepared slides to characterize
cells, tissues, and organs. Slides can be prepared from thin sections of tissues of
interest, as well as from intact single or multi cell cultures. In general, the samples must
be fixed in a medium, such as formalin, that preserves the biological material in its
native state. The sample is often embedded in paraffin or plastic, and a microtome is
used to cut the resultant block into thin sections (typically 4 – 10 m thick), which can be
mounted on glass slides. The embedding material is then removed by solvent
extraction, and the samples can be stained to highlight structural or compositional
features of interest. The sample is then covered in a mounting medium, such as
permount, and a glass cover slip is added to ensure the long-term stability of the
sections. This laboratory demonstration involves the microscopic examination of two
different sets of histological slides: (i) the basic zoology (BZ) set and (ii) the introductory
biology (IB) sets. Demonstration microscopes have been setup with the slides at the
appropriate magnification (10X or 40X) for comparative purposes. Changing to a higher
magnification can result in accidental slide breakage. DO NOT CHANGE THE
OBJECTIVE. If you are having difficulty, please ask for help.
Examine the slides, make drawings and notes and answer the questions. Use
the questions as a guide to the observation you should make. You will need to do
additional reading to completely answer some questions.
Suggested reference: www.microscopyu.com
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Student #: _________________
Questions (3 to Hand in)
Cells
1. Make drawings to compare the frog egg (BZ11) and human sperm (BZ7)
samples. While the oocytes of frogs can be over 2 mm in diameter, human eggs
are much smaller, at approximately 120 m. Would you expect such a
difference? Why or why not? In contrast, human sperms are much smaller, in the
size range of 10 – 15 m. What implications does this have about the roles of the
egg versus the sperm in embryogenesis? If the doubling time for the fertilized
frog egg is 30 minutes, estimate the time at which the sample was fixed.
Tissues
2. What is the function of an artery? What is the function of a vein? Examine the
transverse section of an artery and vein (BZ6), identify and draw the structure of
each type of vessel. (Hint: after histological processing, veins tend to collapse).
Explain the structure-function relationship for the differences between the two
types of vessels.
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3. Examine the transverse and longitudinal sections of the skeletal muscle sample
(BZ2) and draw a three-dimensional schematic of the tissue. From an
engineering perspective, propose how the structure of skeletal muscle enables
muscle contraction and relaxation. Make a drawing to compare the skeletal
muscle and cardiac muscle (IB13) samples. Note that branching is only present
in the cardiac muscle sample.
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