2. Water Microbiology
Purposes
What is water and water microbiology?
Why water should be tested microbiologically?
How water could be sampled ?
How transported ?
3. Different names are given to water's various forms:
1. according to state
• Solid - ice
• Liquid - water
• Gaseous - water vapor
2. levitating particles
• clouds
• Fog
• mist
3. according to occurrence:
• Groundwater
• Fresh water
• Surface water
• Mineral water - contains much minerals
• Brackish water
• Dead water - strange phenomenon which can occur when a layer of fresh or brackish
water rests on top of denser salt water, without the two layers mixing. It is dangerous for
ship traveling.
• Seawater
• Brine
4. 4. according to uses
• tap water
• bottled water
• drinking water
• purified water:
distilled water
double distilled water
deionized water
5. according to other features
• soft water – contains less minerals
• hard water – from underground, contains more minerals
• distilled water, double distilled water, deionized water - contains no minerals
• Water of crystallization — water incorporated into crystalline structures
• Hydrates — water bound into other chemical substances
• heavy water – made from heavy atoms of hydrogen - deuterium. It is in nature in
normal water in very low concentration. It was used in construction of first nuclear
reactors.
6. according to microbiology
• drinking water
• wastewater
• stormwater or surface water
5. Water cycle:
The water cycle (known scientifically as the hydrologic cycle) refers to the continuous
exchange of water within the hydrosphere, between the atmosphere, soil water,
surface water, groundwater, and plants.
Water moves perpetually through each of these regions in the water cycle consisting of
following transfer processes:
1. evaporation from oceans and other water bodies into the air and transpiration from
land plants and animals into air.
2. precipitation, from water vapor condensing from the air and falling to earth or ocean.
3. runoff from the land usually reaching the sea.
6.
7. Water:
• Water is essential for the maintenance of all life on Earth.
• It also acts as the vector for many diseases caused by bacteria,
viruses, protozoa and worms.
• For water to be regarded as potable, i.e. of a quality fit and
safe for drinking, it must be:
1. free from pathogens.
2. it must not contain any other noxious substances such as
chemical hazards including pesticides, insecticides or
herbicides, artificial fertilizers or heavy metal ions.
3.should not have an unpleasant odor or taste.
8. What are water-borne diseases?
1. Among the bacterial infections that are spread by water are:
• cholera
•enteric fevers
•dysentery.
2. Among the viruses are:
•Hepatitis A
•poliovirus cause infections after drinking contaminated water.
3. Among the protozoa are:
•Amoebic dysentery is caused by the protozoan Entamoeba histolytica and is
spread either by drinking contaminated water or by eating food such as fresh
fruit, salad or raw vegetables that have been washed in contaminated water.
• Other protozoal diseases such as those caused by Giardia intestinalis
(Giardia lamblia)
Balantidium coli and Cryptosporidium species are spread in a similar fashion.
4. Among the helminthes are:
Schistosomiasis, also known as bilharzia, is a water-borne infestation caused
by worms of the genus Schistosoma.
9. Water microbiology assist in providing the
answers :
• How do we know that the water used is safe?
• Can we drink it?
• Is a particular beach okay for swimming?
• Can it be used for irrigation?
The answers to these questions are of vital public health importance to all of us.
The Water Microbiology field can assist in providing the answers.
How can I confirm that the water is microbiologically safe to use for the
intended purpose?
10. 1. Total Coliform (TC)
2. Faecal Coliform (FC)
3. Faecal Streptococci (FS)
4. Enterococci
This is achieved by conducting standard microbiological tests to assess the
sanitary quality of water. These tests are designed to determine the
presence/absence of the following indicator organisms:
Indicator Organisms:
The tests results are compared to standards or guideline values that have been
designed to protect human health.
11. Why do we concentrate on testing for these indicator
organisms?
• It is impossible to test for every known pathogenic organism on a
routine basis.
• Indicator bacteria have been studied extensively. Their detection
and enumeration employ simpler and more economic test that can
be performed routinely.
• The presence of such organisms in a water sample suggest that
the water has been compromised by faecal contamination and
that pathogens may be present.
•Indicator is generally isolated and identified easily in laboratory.
•It is less cost effective in the lab.
12. Aquatic Facilities Microbiological Water Sampling Technique
1. General Rules of Sampling
Take extra care to avoid contaminating the sample container and water sample.
Do Not:
• Contaminate the bottle by touching the inside of the bottle.
• Contaminate the bottle lid by touching the inside rim.
• Put the bottle lid on the ground while sampling.
• Rinse the bottle.
• Transport aquatic facility water samples with other water samples, e.g. effluent or
drinking water.
Always:
• Collect microbiological samples before collecting other samples.
• Label the bottle before sampling.
• Discard damaged or contaminated bottles. If in doubt throw it out and take sample in a
new bottle.
• Wash your hands thoroughly before and collecting samples.
13. 2. Labeling :
• Sender reference number
• Site code
• Point of Collection
• (Aquatic Facility Name and pool (ie toddler’s pool)
• Source (ie Pool outlet)
• Date and time of collection
• Transport temperature (4C or ambient)
• Authority or Company Name
14. 1) A student creates a pendulum effect in order to cast
the bucket out as far as possible.
2) A student aims to retrieve the top surface of the water
sample.
3. Sampling Collection Procedure:
15. 4) A student checks to make sure that the water sample
has not been muddied during retrieval.
3) The student uses his arm like a crane to retrieve the
sample bucket without picking up any bottom sediment.
16. 4. Sample Transportation:
• Temperature :
Once water samples for bacteria are collected, they should be immediately stored within
a chilled insulation container (esky) preferably at a temperature between 1°C and 4°C.
To chill the samples/container, use freezer ice bricks if available, or loose ice.
The chilled temperatures are used to prevent the multiplication of bacteria which
may result in false bacterial counts. Cool and dark conditions should also be
maintained throughout transportation to the laboratory.
• Time:
1. the aim of delivering the samples to the laboratory as soon as possible, or within 6
hours of commencing sampling, whilst keeping the sample bottle temperatures at 4°C
±2°C.
2. Under exceptional circumstances (regional locations), the sampling and transport time
may exceed 6 hours but should never exceed more than 24 hours.
17. 5. Submitting Samples :
• Parameters:
All water test reports list the water quality parameters that were tested. The list includes
only those you asked the laboratory to analyze or the lab recommended for your water
sample.
• The number of parameters can vary from just a few to dozens of tests.
• Results:
The most important information on your water test report are the actual results that the
laboratory found for your water sample.
The result for each test should be compared to the drinking water standard:
1. maximum contaminant level (MCL) for that parameter.
2. Sometimes, the lab reports a water test result as ―ND‖ (Not Detected), which means
the lab was unable to detect any of that contaminant with its equipment.
3. Similarly, some results may have a less-than sign (<) in front of a number.
• Units
Concentrations of contaminants are usually measured in water by a unit of weight such
as milligrams per liter (mg/L), or by a number, for example, number of bacteria per
100 milliliters of water (#/100 ml).
• Standards:
This allows for an easy comparison of your result with the safe or recommended
maximum level for each test parameter.
18. 6. Comments:
Some water testing laboratories will include a brief explanation of your water test results.
• It is often list those contaminants that did not meet the drinking water standard.
• Occasionally, these comments also describe the potential harmful effects of
contaminants that exceed the standard and how to remove these contaminants from the
water.
•Some laboratories, however, do not provide comments, so you need to review the
results yourself. Do not rely on the laboratory to point out important information.
21. Measurements of bacteria
1. Plate Count:
a. Spread (Streak) Plate
b. Pour Plate
2. Direct Observation on Slides
- Petroff-Hausser Chamber Slide
3. Most Probable Number (MPN)
4. Filtration (Membrane filter technique)
30. Petroff-Hausser Counting Chamber
Measurement of Microbial Growth - Measurement of cell
numbers - Direct microscopic counts
• Inexpensive
• Relatively quick
• Gives information about size and morphology
31. Direct microscopic count
In direct microscopic counting: 1) dead cells are not distinguished from
living cells; 2) small cells are difficult to see under the microscope; 3)
precision is difficult to achieve; 4) we need a phase contrast microscope; 5)
not a good method for cell suspensions of low density.
32. There Are More Accurate Methods to
Determine Turbidity Levels
33. Materials per Student
• microscope
• culture of P. aerugenosa, Escherichia coli, bacillus
• lens paper and lens cleaner
• immersion oil
• clean depression slides and coverslips
• petroleum jelly (Vaseline)
• inoculating loop
• toothpicks
• Bunsen burner
34. Principles
Many bacteria show no motion and are termed non-motile. However, in an
aqueous environment, these same bacteria appear to be moving erratically.
This erratic movement is due to Brownian movement.
1. Brownian movement: results from the random motion of the water
molecules bombarding the bacteria and causing them to move (i.e.: vibration
of water molecules). Like Sarcina sp.
2. Gliding motility: Helical-shaped spirochetes have axial fibrils (modified
flagella that wrap around the bacterium) that form axial filaments. These
spirochetes move in a corkscrew- and bending-type motion. (simply slide
over moist surfaces in a form of gliding motion) Treponema pallidum
3. Swimming motility: True motility (self-propulsion) has been recognized in
other bacteria and involves several different mechanisms. Bacteria that
possess flagella exhibit flagellar motion.
4. Tumbling motility: Like in Listeria monocytogenes
The above types of motility or non motility can be observed over a long period
in a hanging drop slide. Hanging drop slides are also useful in observing the
general shape of living bacteria and the arrangement of bacterial cells
when they associate together.
36. Procedure:
1. With a toothpick, spread a small ring of Vaseline around the concavity of a
depression slide. Do not use too much Vaseline.
2. After thoroughly mixing one of the cultures, use the inoculating loop to
aseptically place a small drop of one of the bacterial suspensions in the center
of a covers lip.
3. Lower the depression slide, with the concavity facing down, onto the covers
lip so that the drop protrudes into the center of the concavity of the slide. Press
gently to form a seal.
4. Turn the hanging drop slide over and place on the stage of the microscope
so that the drop is over the light hole.
5. Examine the drop by first locating its edge under low power and focusing on
the drop. Switch to the high-dry objective and then, using immersion oil, to the
90 to 100× objective. In order to see the bacteria clearly, close the diaphragm
as much as possible for increased contrast.
Note bacterial shape, size, arrangement, and motility. Be careful to distinguish
between motility and Brownian movement.
6. Discard your covers lips and any contaminated slides in a container with
disinfectant solution.
37.
38. Bacteria
1. Aeromonas in Finished Water by Membrane Filtration
2. Total Coliforms and Escherichia coli in Water by Membrane Filtration Using a
Simultaneous Detection Technique
3. Escherichia coli (E.coli) in Water by Membrane Filtration Using Modified
membrane-Thermotolerant Escherichia coli Agar
4. Enterococci in Water by Membrane Filtration Using membrane-Enterococcus
Indoxyl-ß-D-Glucoside Agar
5. Enterococci in Water by Membrane Filtration Using membrane-
Enterococcus-Esculin Iron Agar
6. Escherichia coli (E. coli) in Water by Membrane Filtration Using membrane-
Thermotolerant Escherichia coli Agar
7. Improved Enumeration Methods for the Recreational Water Quality
Indicators: Enterococci and Escherichia coli (March 2000)
39. •Direct measurements of microbial growth: total and viable counts.
•Advantages and disadvantages.
a) Direct microscopic count
b) Viable cell counting
a. Direct microscopic count:
on samples dried on slides or on samples in liquid using growth chambers.
41. Bacteriological Analysis of Water
The principal means through which pathogenic microorganisms reach water supplies is fecal
contamination. The method for bacteriologic examination of water is designed to provide an
index of fecal contamination. Pathogenic microorganisms do not necessarily multiply in
water, and therefore they may be present in small numbers that are difficult to demonstrate in
culture. Escherichia coli, other coliform bacteria, and enterococci, however, are not only
abundant in feces but also usually multiply in water, so that they are present in large, readily
detectable numbers if fecal contamination has occurred. Thus, culture demonstration of E. coli
and enterococci in water indicates a fecal source of the organisms. In water from sources
subjected to purification processes (such as reservoirs), the presence of E. coli or enterococci
may mean that chlorination is inadequate. By bacteriologic standards, water for drinking (i.e.,
potable water) should be free of coliform and enterococci.
The term “coliform,” which refers to lactose-fermenting gram negative enteric bacilli, is now
obsolete except in sanitary bacteriology.
42. There are two principal groups of coliform bacteria: the fecal coliform
(which includes the bacterium Escherichia coli has been most studied)
and the total coliform group, which includes the fecal coliform and
consists mainly of species from the genera Citrobacter, Enterobacter,
Escherichia, and Klebsiella. The former are exclusively fecal in origin,
whereas the latter, although commonly found in feces, also occur
naturally in Soils and waters. Only the fecal coliform is definitive indicators of
fecal pollution.
In water bacteriology the total coliform are regarded as "presumptive"
indicators of pollution but in Wastewater bacteriology, this group is
considerably less importance, because many of them are non-fecal in origin
and they can multiply in the environment under suitable conditions,
especially in hot climates. Thus their presence or numbers may not necessarily
relate to either the occurrence or degree of fecal pollution
43. 1. Total Coliform (TC)
2. Faecal Coliform (FC)
3. Faecal Streptococci (FS)
4. Enterococci
These tests are designed to determine the presence/absence of the following
indicator organisms:
Indicator Organisms:
The tests results are compared to standards or guideline values that have been
designed to protect human health.
44. Principles
• The number of total coliform (Enterobacter, Klebsiella, Citrobacter, Escherichia) in a
water sample can be determined by a statistical estimation called the most probable
number (MPN) test.
• This test involves a multiple series of Durham fermentation tubes and is divided into
three parts: the presumptive, confirmed, and completed tests.
A presumptive test for coliform is performed by inoculating a sample of water into
tubes of lactose broth containing Durham tubes. After 24 to 48 hours of incubation at
35°C, the tubes are examined for the presence of acid and gas as an indication of lactose
fermentation. Other than coliform, few organisms found in water can ferment lactose
rapidly with production of gas.
Gaseous fermentation of lactose within 24 to 48 hours provides presumptive evidence
of the presence of coliform. The test must be confirmed, however, to exclude the
possibility that another type of organism provided the positive lactose result.
45. Confirmed test
Is done by plating a sample of the positive lactose broth culture into a lactose
broth again and same result which may provide confirmation of the
presumptive test.
Inoculating in peptone water at 42 °C or brilliant green lactose bile broth.
Completed test
Growing on differential agar medium. Eosin methylene blue (EMB) agar is
frequently used. Coliform colonies ferment the lactose of EMB and consequently
have a deep purple color with a coppery, metallic sheen
Requires inoculation of another lactose broth and an agar slant with isolated
colonies from EMB. Gas formation in the lactose broth and microscopic
demonstration of gram-negative, non-spore former rods on the agar slant are
considered complete evidence of the presence of coliform organisms in the
original sample.
46. Learning Objectives
Each student should be able to
1. Determine the presence of coliform bacteria in a water sample
2. Obtain some index as to the possible number of coliform bacteria present in the water
sample being tested
3. List and explain each step (presumptive, confirmed, completed) in the multiple tube
technique for determining coliforms in the water sample
4. Perform the presence-absence coliform test MPN
Coliform Guide
• Citrobacter
• Escherichia
• Enterobacter
• Klebsiella
47. Materials per Group of Students:
• (10) 10-ml single-strength lactose broth (SSLB) in Durham fermentation tubes (lauryl tryptose
broth or lactose broth)
• (5 )10-ml double-strength lactose broth (DSLB) in Durham fermentation tubes
• 125-ml water sample (each group of students should bring in their own from a possible
contaminated water system) at room temperature. (If the water samples are collected early, they
should be refrigerated until analyzed.)
• petri plate containing Levine’s EMB agar (or LES Endo agar)
• tryptic agar slant
• 3 tubes brilliant green lactose bile broth (brilliant green bile broth 2%) or 2 tubes lauryl tryptose
broth containing Durham tubes
• 1 sterile 10-ml pipette with pipettor
• 2 sterile 1-ml pipettes
• wax pencil
• test-tube rack
• 35°C incubator
• inoculating loop and needle
• Bunsen burner
48. Procedure for the MPN Test
First Period
•Presumptive Test
1. Mix the bottle of water to be tested 25 times. Inoculate five of the double-strength
lactose (or lauryl tryptose) broth tubes with 10 ml of the water sample; five single-
strength tubes with 1 ml of the water sample; and five single-strength tubes with 0.1 ml
of the water sample. Carefully mix the contents of each tube without spilling any of the
broth by rolling the tubes between the palms of your hands. Using the wax pencil, label
all tubes with your (name, date, and the amount of water added).
2. Incubate the three sets of tubes for 24 to 48 hours at 35°C.
3. Observe after 24 ±2 and 48 ±3 hours. The presence of gas in any tube after 24 hours
is a positive presumptive test. The formation of gas during the next 24 hours is a
doubtful test. The absence of gas after 48 hours is a negative test.
4. Determine the number of coliforms per 100 ml of water sample. For example, if gas
was present in all five of the 10-ml tubes, only in one of the 1-ml series, and none in the
0.1-ml series, your test results would read 5–1–0. Table indicates that the MPN for this
reading would be 33 coliforms per 100 ml of water sample.
49.
50. Most Probable Number (MPN)
These are gas-filled
tubes, an indication
of bacterial growth
(fermentation).
Looking for
sufficient dilution
that ~half of tubes
show growth.
Reciprocal of
that dilution
bacterial
density.
51. • Second Period
Confirmed Test
1. Record your results of the presumptive test in the report for exercise 46.
2. Using an inoculating loop, from the tube that has the highest dilution of water sample
and shows gas production transfer one loopful of culture to the brilliant-green lactose
bile broth tube. Incubate for 48 ± 3 hours at 35°C. The formation of gas at any time
within 48 hours constitutes a positive confirmed test.
52. Third Period
Completed Test
1. Record your results of the confirmed test in the report.
2. From the positive brilliant green lactose bile broth tube, streak a LES Endo or
Levine’s EMB plate.
3. Incubate the plate inverted for 24 hours at 35°C.
4. If coliforms are present, select a well-isolated colony and inoculate a single-strength,
brilliant green lactose bile broth tube and streak a nutrient agar slant.
5. Gram stain any bacteria found on the slant.
6. The formation of gas in the lactose broth and the demonstration of gram-negative,
nonsporing rods in the agar culture is a satisfactorily completed test revealing the
presence of coliforms and indicating that the water sample was polluted. This is a
positive completed test.
53.
54. Questions:
•Write scientific interpretations for your results, and final comments?
•Why we depended on gas production rather than acid formation?
•What is the bacteriological standard for potable water?
•Why the bacteriological analysis of water depends on recognition of
coliform and enterococci instead of direct detection of pathogenic
bacteria?
•Why the positive presumptive tests of water must be confirmed?
•List at least three waterborne infectious diseases (bacterial name).
•Compare between (water samples), and (which method was better among
them).
•Compare between (Fecal coliform and Total coliform)
55.
56. Principles for Membrane Filter Technique:
• This technique involves filtering a known volume (100 ml for drinking water
samples) of water through a special sterile filter.
• These filters are made of nitrocellulose acetate or polycarbonate, are 150 μ
thick, and have 0.45 μ diameter pores. A grid pattern is typically printed on
these filter disks in order to facilitate colony counting.
• When the water sample is filtered, bacteria (larger than 0.45 μ) in the sample
are trapped on the surface of the filter. The filter is then carefully removed,
placed in a sterile Petri plate on a pad saturated with a liquid or agar-based
medium, and incubated for 20 to 22 hours at 35°C.
• it is assumed 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.
• Fecal streptococci are the Lancefield Group D streptococci that occur in the
feces of humans and other warm-blooded mammals.
57. • Total coliform colonies will be pink to dark red in color and will appear to
have a golden green metallic sheen. Fecal coliform colonies will appear blue,
and fecal streptococci colonies will appear light pink and flat, or dark red.
• In determining total coliform, the amount of water filtered should be enough to
result in the growth of about 20 to 80 colonies and no more than a total of 200
bacterial colonies of all types. About 50 to 200 ml of unpolluted water is often
adequate for such bacterial counts. Polluted water may contain so many
coliform that it will be necessary to dilute 1 ml or less of sample with about 50
ml of sterile water. This is done in order to provide enough volume for uniform
bacterial dispersion across the filter surface, in addition to providing an
appropriately low coliform count.
• Coliform density is expressed in terms of the number of coliform per 100 ml of
water and is calculated according to the following formula:
58. • The number of coliform should be given to show two significant figures
per 100 ml.
•The standard set for potable (drinking) water is a limit of 1 coliform per 100 ml
and an action limit of 4 coliform per 100 ml. An action limit means that the
water company or other provider must take immediate action to remedy the
problem(s) that is/are responsible for the presence of coliform.
• From positive fecal coliform and fecal streptococci test results, one can be
fairly certain that the water pollution is from which fecal source. However, in
order to determine whether the fecal source is from human or animal feces,
Public Health authorities rely on a ratio expressed as:
59. 1. Those samples showing a higher fecal coliform count than fecal strep count are
likely to contain wastes from humans. In most cases, the FC/FS ratio will be greater
than 2. When the ratio is equal to or greater than 4, one can be assured that the
pollution is from human fecal material.
2. When it shows a higher fecal strep count than fecal coliform count, it is most likely
that the pollution is from animal origin.
3. If ratios fall in between two and four, estimates must be made as to how close the
ratio is to either the human or animal value. The following table shows some typical
FC/FS ratios:
60. :Total Coliform Test
1. Using the sterile forceps, place a sterile absorbent pad into each of three
petri plates. With the wax pencil, label these plates with your name, date,
and TCT (total coliform test).
2. Add 2.0 ml of M-Endo broth MF (or m-ColiBlue 24 broth) to the surface of
each pad.
3. Filter 1, 5, and 15 ml of the water sample, and add the membranes
respectively to each plate.
4. Incubate the plates at 35°C for 22 to 24 hours.
5. Count only those colonies that are pink to dark red with a metallic sheen. Use
a plate containing 20 to 80 colonies and no more than 200 of all types of
colonies. If the m-ColiBlue 24 broth is used, blue to purple colonies indicate
E. coli. The total coliform count is given by the sum of red and blue colonies.
6. Record your results in the report.
61. Fecal Coliform Test:
1. Using the sterile forceps, aseptically insert sterile pads into three snap-lid
petri plates. Using the wax pencil, label these plates with your name, date,
and FCT (fecal coliform test).
2. Add 2.0 ml of M-FC broth to the surface of each pad.
3. Filter 1, 5, and 15 ml of the water sample, and add the membranes
respectively to each plate.
4. Snap the lids of the Petri plates, seal them with waterproof tape, and place
them in a Whirl pack bag.
5. Incubate the plates in a 44.5° ±0.2°C water bath for 22 to 26 hours. Make
sure the bags are beneath the surface.
6. Count only blue-colored colonies on a plate containing 20 to 60 fecal
coliform colonies.
7. Record your results in the report.
62. Fecal Streptococcus Test:
1. Aseptically insert a sterile absorbent pad into each of three Petri plates.
Using the wax pencil, label these plates with your name, date, and FST
(fecal streptococcus test).
2. Add 2 ml of KF streptococcus agar; allow the agar to cool.
3. Filter water sample volumes of 1, 5, and 15 ml as per previous test. Place the
membranes in the labeled Petri plates.
4. Incubate these plates for 48 hours at 35°C.
5. Count only those colonies that are light pink and flat, or smooth dark red
ones with or without pink margins. Use the plate containing 20 to 100
colonies.
6. Record the number of colonies in the report.
63.
64.
65. Membrane Filter Technique.
(a) Total Coliform test: Total coliform on a membrane filter (M-Endo MF broth medium).
Notice the dark red to purple colonies with a metallic sheen.
(b) Fecal Coliform Test: Fecal coliform on a membrane filter (M-FC broth medium).
Notice the blue-colored colonies.
66. (c) Fecal Streptococcus Test: Fecal streptococci on KF streptococcus agar. Notice the
light pink colonies.
(d) HACH m-ColiBlue 24 Broth: The sum of red and blue colonies indicates total
coliform, while the blue specifies E. coli.
67. HINTS AND PRECAUTIONS:
(1)The broth media should be freshly prepared on the day of the exercise.
(2) Water should not be used that is high in turbidity or contains a lot of algae.
Coliform density is always expressed in terms of a 100-ml water sample. If
the water sample is diluted, the number of colonies must still be calculated
for a 100-ml sample. Similarly, if less than 100 ml of water is filtered, the
coliform density must still be expressed in terms of 100 ml.
(3) If the water sample will not be tested immediately, store it in the refrigerator
to prevent extra microbial growth.
(4) When using the membrane filter technique, the 19th edition of Standard
Methods for the Examination of Water and Wastewater suggests that the
following sample volumes be used for total coliform tests:
75. Are inactive molecules outside of the host cell
and active only inside host cells.
Basic structure consists of protein shell (capsid)
surrounding nucleic acid core.
Nucleic acid can be either DNA or RNA but not
both.
Nucleic acid can be double-stranded DNA,
single-stranded DNA, single-stranded RNA, or
double-stranded RNA.
76.
77. Poxvirus, DNA virus Mumps virus RNA Herpesvirus DNA
Rhabdovirus HIV (AIDS)
RNA RNA
Bacteriophage Papillomavirus
a DNA virus
Adenovirus
a DNA virus
78. Phage Multiplication
Lysogeny a form of bacteriophage replication in which the viral
genome is integrated into that of the host and is replicated along
with it.
Lytic cycle a process of viral replication involving the bursting of
the host cell and release of new viral particles.
Viruses which infect bacteria are known as bacteriophage, and
those which infect coliform bacteria are called coliphage.
The phages of coliform bacteria are found anywhere coliform
bacteria are found.
Concentrations of human viruses in raw sewage range from (103–
107 /L). Concentration of coliphage in raw sewage ranges from (10
to 100 /ml).
79. There are many potential applications of bacteriophages as
environmental indicators. These include use as indicators of
sewage contamination, efficiency of water and wastewater
treatment, and survival of enteric viruses and bacteria in
the environment. The use of bacteriophages as indicators of the
presence and behavior of enteric bacteria and animal viruses
has always been attractive because of the ease of detection
and low cost associated with phage assays.
In addition, they can be quantified in environmental samples
within 24 hours as compared to days or weeks for enteric
viruses. Coliphage have been the most commonly used in this
context although other bacteriophages and cyanophage (i.e.,
viruses of blue-green algae) have also been studied. Much of
the justification for the study of coliphage behavior in nature
has been to gain insight into the fate of human pathogenic
enteric viruses. As a result, more is probably known about the
ecology of coliphage than any other bacteriophage group.
80. Coliphage in water are assayed by addition of a sample to soft or
overlay agar along with a culture of E. coli in the log phase of growth.
The phages attach to the bacterial cell and lyses the bacteria. The
bacteria produce a confluent lawn of growth except for areas where
the phage has grown and lyses the bacteria. These resulting clear
areas are known as plaques. A soft agar overlay is used to enhance
the physical spread of the viruses between bacterial cells.
To obtain optimal plaque formation it is important that the host
bacteria are in the log phase of growth. This ensures that all the
phage attach to live bacteria and produce progeny. This requires that
a culture of host bacteria is prepared each day that an assay is
performed. Usually, a culture is incubated the day before the assay to
obtain a culture in the stationary phase. This is used to inoculate a
broth which is incubated to obtain enough host bacteria in the log
phase for the assay (this usually requires 2–3 hours of incubation in a
shaking water bath at 35 to 37°C).
81. MANY VIRUSES CAUSE DISEASE IN ANIMALS
Viruses that infect animal cells cause diseases.
RNA viruses have RNA as their genetic material and
responsible for flu, cold, measles, mumps, AIDS, polio.
DNA viruses have DNA as their genetic material and cause
hepatitis, chicken pox, herpes.
82. VIRAL DNA MAY BECOME PART OF HOST CHROMOSOME
Viruses are packaged genes-can only reproduce inside
cells.
Lytic cycle-viral replication cycle resulting in the
release of new viruses by Lysis of host cell.
Lysogeny cycle-a bacteriophage replication cycle in
which the viral genome is incorporated into the
bacterial host chromosome and the host cell is not lyses
unless the viral genome leaves the host chromosome.
84. The Lysogeny state in bacteria.
- A bacterial DNA molecule can
accept and insert viral DNA
molecules at specific sites on its
genome.
-This additional viral DNA is
duplicated along with the
regular genome and can
provide adaptive
characteristics for
the host bacterium
85.
86.
87.
88.
89. Precautions:
When collecting sewage samples always wear latex
gloves. Raw sewage is a potent source of bacterial,
viral, and fungal pathogens.
Raw sewage rich in bacteriophage is best collected at
municipal sewage treatment plants. Usually,
collection is made through manhole access.
As emphasized in previous points of this manual,
using pipette inhibited!
90. 1. DO:
Bacteria must be in the log phase of growth for optimal phage plaque formation. This means
that a new culture must be grown under a defined set of conditions (temperature, shaking or
non-shaking) each time.
Be sure to shake the tube containing overlay agar to get as much out of the tube as possible.
2. DO NOT:
Do not allow the bacteria and phage to set in the water bath too long (no more than 1–2
minutes) or they will be killed by the heat.
Do not allow the molten agar to set in the 45°C water bath for more than 1–2 hours as the
water evaporates causing lumps of agar to form.
Potential hazards:
Remember if you are handling sewage, it may contain pathogens.
Handle with care.
93. Pollution categories
1. Point source pollution
refers to contaminants that enter a waterway through a
discrete conveyance, such as a pipe or ditch. Examples of
sources in this category include discharges from a sewage
treatment plant, a factory, or a city storm drain.
2. Non-point source pollution
refers to diffuse contamination that does not originate from a
single discrete source. As rainfall runs over the surface of
roofs and the ground, it may pick up various contaminants
including soil particles and other sediment, heavy metals,
organic compounds, animal waste, and oil and grease.
Some jurisdictions require storm water to receive some
level of treatment before being discharged directly into
waterways.
94. Causes of water pollution
The specific contaminants leading to pollution in water include:
1. Pathogens
Coliform bacteria are a commonly-used bacterial indicator
of water pollution, although not an actual cause of disease.
Other microorganisms sometimes found in surface waters
which have caused human health problems include:
Cryptosporidium parvum
Giardia lamblia
Salmonella
Novovirus and other viruses
Parasitic worms (helminthes).
95. Water used for drinking and cooking should be free of
pathogenic (disease causing) microorganisms that cause such
illnesses as typhoid fever, dysentery, cholera, and
gastroenteritis. Whether a person contacts these diseases
from water depends on:
Type of pathogen
Number of organisms in the water (density)
Strength of the organism (virulence)
Volume of water ingested
Susceptibility of the individual.
Purification of drinking water containing pathogenic
microorganisms requires specific treatment called disinfection.
96. 2. Chemical and other contaminants:
a. Organic water pollutants include:
• Detergents
• Disinfection by-products such as chloroform
• Food processing waste.
• Insecticides and herbicides.
• Petroleum hydrocarbons: fuels (gasoline, fuel oil) and lubricants (motor oil).
• Volatile organic compounds (VOCs), such as industrial solvents, from improper
storage.
b. Inorganic water pollutants include:
Acidity caused by industrial discharges (especially sulfur dioxide).
Ammonia from food processing waste.
Chemical waste as industrial by-products.
Fertilizers containing nutrients--nitrates and phosphates--which are found in storm
water runoff from agriculture, as well as commercial and residential use.
Heavy metals from motor vehicles.
97. 3. Physical contaminants:
Thermal pollution: Thermal pollution is the rise or fall in
the temperature of a natural body of water caused by
human influence. A common cause of thermal pollution is
the use of water as a coolant for industrial manufacturers.
Macroscopic pollution--large visible items polluting the
water--may be termed "floatables":
Trash (e.g. paper, plastic, or food waste)
Shipwrecks, large derelict ships
98. According to a 2007 World Health Organization report, 1.1
billion people lack access to an improved drinking water
supply.
88% of the 4 billion annual cases of diarrheal disease are
attributed to unsafe water and inadequate sanitation and
hygiene, and 1.8 million people die from diarrheal diseases
each year.
The WHO estimates that 94% of these diarrheal cases are
preventable through modifications to the environment,
including access to safe water. Simple techniques for treating
water at home, such as chlorination, filters, and solar
disinfection, and storing it in safe containers could save a
huge number of lives each year.
99. Sewage treatment
Is the process of removing contaminants from wastewater. It includes physical,
chemical and biological processes to remove physical, chemical and biological
contaminants.
Sampling: Sampling of water for physical or chemical testing can be done by
several methods, depending on the accuracy needed and the characteristics of
the contaminant.
1. Physical testing:
Temperature, solids concentration and turbidity.
2. Chemical testing:
Analytical chemistry. Many published test methods are available for both organic
and inorganic compounds. Frequently-used methods include pH,
biochemical oxygen demand (BOD), chemical oxygen demand (COD),
nutrients (nitrate and phosphorus compounds), metals (including copper,
zinc, cadmium, lead and mercury), oil and grease, total petroleum
hydrocarbons (TPH), and pesticides.
3- Biological testing:
plant, animal, and/or microbial indicators
100. Treatment steps
1. Primary treatment:
Can physically remove 20-30% of the BOD that is present in
particulate form, in this treatment particulate material is
removed by screening, precipitation of small particulates by
addition of alum and other coagulation agents, and settling in
tanks. The resulting solid material is usually called (Sludge).
2. Secondary treatment:
is used after primary treatment for the biological removal of
dissolved organic matter, about 90-95% of the BOD and many
bacterial pathogens are removed by this process. Under aerobic
condition, dissolved organic matter will be transformed into
additional microbial biomass plus carbon dioxide. Minerals in
water also may be tied up in microbial biomass. When microbial
growth is completed, under ideal conditions the microorganisms
will be aggregate and form a stable floc structure.
101. 3. Tertiary treatment
The purpose of tertiary treatment is to provide a final treatment stage to raise the effluent
quality before it is discharged to the receiving environment (sea, river, lake, ground, etc.).
More than one tertiary treatment process may be used at any treatment plant. If
disinfection is practiced, it is always the final process. It is also called "effluent polishing".
Filtration
Sand filtration removes much of the residual suspended matter. Filtration over activated
carbon removes residual toxins.
Disinfection
The purpose of disinfection in the treatment of wastewater is to substantially reduce the
number of microorganisms in the water to be discharged back into the environment.
The effectiveness of disinfection depends on the quality of the water being treated (e.g.,
cloudiness, pH, etc.), the type of disinfection being used, the disinfectant dosage
(concentration and time), and other environmental variables. Cloudy water will be
treated less successfully since solid matter can shield organisms, especially from
ultraviolet light or if contact times are low. Generally, short contact times, low doses and
high flows all militate against effective disinfection. Common methods of disinfection
include ozone, chlorine, or ultraviolet light. Chloramines, which is used for drinking
water, is not used in wastewater treatment because of its persistence.
106. Sources of water
1. Groundwater
2. Upland lakes and reservoirs
3. Rivers, canals and low land reservoirs
4. Atmospheric water generation
5. Rainwater harvesting or fog collection
6. Desalination of seawater by distillation or reverse
osmosis
107. Purification steps
1. Screening
Removing of large particle or macroscopic contaminant such
as bottle and paper …etc.
2. Coagulation: In rapid mixer
Flocculation: is a process which clarifies the water. Clarifying
means removing any turbidity or color so that the water is
clear and colorless.
Coagulants / flocculating agents that may include:
1. Aluminum sulphate.
2.PolyDADMAC is an artificially produced polymer
108. 3. Sedimentation:
Waters exiting the flocculation basin may enter the sedimentation
basin, also called a clarifier or settling basin.
109. It is a large tank with slow flow, allowing floc to settle to the bottom. The sedimentation
basin is best located close to the flocculation basin so the transit between does not
permit settlement or floc break up.
110.
111. 4. Filtration:
After separating most floc, the water is filtered as the final step to remove remaining suspended
particles and unsettled floc.
112. 5. Disinfection (Chlorination) :
Disinfection is accomplished both by filtering out harmful microbes and also by adding
disinfectant chemicals in the last step in purifying drinking water. Water is disinfected to kill
any pathogens which pass through the filters. Possible pathogens include viruses, bacteria,
including Escherichia coli, Campylobacter and Shigella, and protozoa, including Giardia
lamblia and other cryptosporidia.
113.
114. Aim of lecture
What is disinfectant?
Types of disinfectant
Mechanism of each method
What is the health effects of its bye-product
How can MIC be determined?
How differentiate between MIC and MBC?
How determine exposure time of disinfectant ?
115. Disinfection mechanism
o Cell wall corrosion in the cells of microorganisms.
o Changes in cell permeability.
o Protoplasm or enzyme activity (because of a structural
change in enzymes).
o Change in essential structure of cell (DNA)
These disturbances in cell activity cause microorganisms to:
I. Unable to multiply. This will cause the microorganisms to
die out.
II. Oxidizing disinfectants also demolish organic matter in
the water, causing a lack of nutrients.
117. I. Chlorination
In chlorination, chlorine gas, sodium or calcium hypochlorite is added to
water. A minimum reaction period of 20 minutes is also required for
effective water disinfection.
Why is Chlorine Added to Tap Water?
Chlorination is effective against many pathogenic bacteria, but at
normal dosage rates it does not kill all viruses, cysts, or worms.
When combined with filtration, chlorination is an excellent way to
disinfect drinking water supplies.
Factors affecting chlorine efficiency: Interaction between chlorine and
the microorganisms results in an effective disinfection process:
Contact time varies with chlorine concentration.
The type of pathogens present and dose
pH
Temperature of the water.
118. The Benefits of Chlorine:
Potent Germicide
Taste and Odor Control
Chlorine disinfectants reduce many disagreeable tastes and odors. Chlorine
oxidizes is naturally occurring substances such as foul-smelling algae
secretions, sulfides and odors from decaying vegetation.
Chemical Control
Chlorine disinfectants destroy hydrogen sulfide (which has a rotten egg odor)
and remove ammonia and other nitrogenous compounds that have
unpleasant tastes . They also help to remove iron and manganese from raw
water.
Biological Growth Control
Chlorine disinfectants eliminate slime bacteria, molds and algae that
commonly grow in water supply
119. How Chlorine Kills Pathogen:
Upon adding chlorine to water, two chemical species, known
together as “free chlorine,” are formed. These species,
hypochlorous acid (HOCl, electrically neutral) and
hypochlorite ion (OCl-, electrically negative), behave very
differently.
Hypochlorous acid is not only more reactive than the
hypochlorite ion, but is also a stronger disinfectant and oxidant.
The ratio of hypochlorous acid to hypochlorite ion in water is
determined by the pH.
At low pH (higher acidity), hypochlorous acid dominates.
High pH hypochlorite ion dominates.
120. 2- Chlorine Dioxide:
Chlorine Dioxide is an chemical which is used for water
disinfection, which replaces chlorine in more and
more applications due to its multiple advantages:
Its disinfection force is stronger and independent
upon water’s pH value. Due to its specific chemistry,
no by-products can develop.
The much longer half-life affords better depot action
in treated water.
In opposition to chlorine, Chlorine Dioxide is able to
remove biofilm in pipe systems and tanks to abolish
growth of Legionella.
121. What are the health effects of chlorine by-products?
Chloroform
Bromodichloromethane
Chlorodibromomethane
Chloroacetic acid
Dichloroacetic acid
Trichloroacetic acid
Dichloroacetonitrile
122. 3- Ozonisation
Ozone is unstable gas comprising of three oxygen atoms, the gas
will readily degrade back to oxygen, and during this transition a
free oxygen atom, or radical is form. Ozone has a negative
charge, and upon reaction, the particles are neutralized and will
precipitate.
Ozone has greater disinfection effectiveness against bacteria
and viruses compared to chlorination.
1. Oxidizing properties
2. Reduce the concentration of iron, manganese, sulfur
3. Reduce and eliminate taste and odor problems.
Ozone is unstable, and it will degrade over a time frame ranging
from a few seconds to 30 minutes. The rate of degradation is a
function of water chemistry, pH and water temperature.
123.
124. How Ozone Kills Pathogen:
The free oxygen radical is highly reactive and short
lived, under normal conditions it will only survive for milli
seconds, and during this time frame it will oxidize virtually
any chemical species.
For example it will oxidize iron, manganese and may
other heavy metals, it will crack the carbon double
bond of organic molecules such as dissolved proteins. It
will also oxidize the proteins in the cell wall of
bacteria, the cilia of protozoa, the shell of virus. In
fact ozone has 7 times oxidizing capacity of free
chlorine, also does not produce any toxic residuals.
125. 4. UV radiation disinfection mechanisms
is based on a physical phenomenon whereby short wave UV
radiation acts on the genetic material (DNA) of the microorganisms
and the viruses, destroying them rapidly without producing any major
physical or chemical changes in the treated water.
UV inactivation is thought to occur as a result of the direct
absorption by the microorganism of the UV radiation, bringing about
an intracellular photochemical reaction that change the biochemical
structure of the molecules (probably of the nucleic acids) that are
essential to the microorganism’s survival. It has been shown that
irrespective of the duration and intensity of the dosage, the
expending of the same total energy will result in the same degree of
disinfection.
The ultraviolet radiation system often include activated carbon
filter to remove metals and particulates. Effectiveness of an ultraviolet
radiation system depends on the intensity of the lamp. The
minimum dose of UV light to inactivate bacteria is 38 mWs/cm2 set by
the NSF International.
126. Properties of ultraviolet radiation
UV wavelengths are very similar to those of sunlight. The most important
parameters of UV radiation relating to water disinfection are:
Wavelength: The germicidal portion is between 240 and 280 nm
(nanometers) with maximum disinfecting efficiency existing at close to 260
nm.
Condition of the water
Intensity of radiation
Type of microorganisms
Exposure time
Municipal potable water supplies are usually chlorinated to provide a residual
concentration of 0.5 to 2.0 ppm. Here, in Kurdistan, the recommended
concentrations for water disinfection are 0.7 ppm and 1.7 ppm (in the case of
epidemic water born disease).
127. 1) Estimation of MIC
1. Place two sets of nine sterile tubes in a rack and label them set for
Chlorine (Cl2) and set for Chlorine dioxide (ClO2).
2. With a 5-ml pipette add 2 ml of sterile broth to each tube.
3. Add 2 ml of the Chlorine (Cl2) and Chlorine dioxide (ClO2) to
the first tube of each sets of. Discard the pipette. The
concentration of chlorine and chlorine dioxide in the first tube is
(2ppm and 1.5ppm) respectively.
4. Take a fresh pipette, introduce it into the first tube (chlorine:
2ppm and Chlorine dioxide: 1.5 ppm), mix the contents
thoroughly, and transfer 2 ml from this tube into the second tube
(chlorine: 1ppm and Chlorine dioxide: 0.75ppm). Discard the
pipette.
5. With a fresh pipette, mix the contents of the second tube and
transfer 2 ml to the third tube (chlorine: 0.5ppm and Chlorine
dioxide: 0.38ppm). Discard the pipette.
6. Continue the dilution process through tube number 7. The
eighth and ninth tubes receive no chlorine and chlorine dioxide.
128. 7. After the contents of the seventh tube are mixed, discard 2 ml of broth so that
the final volume in all tubes is 2 ml.
8. From the plate culture of E. coli prepare a suspension of the organism in 5 ml of
(chlorine free distilled water) equivalent to a McFarland 0.5 standard.
9. With a fresh pipette, mix the contents of the tube well. Add 0.1 ml of this E. coli
suspension to the chlorine and chlorine dioxide containing broth tubes 1
through 7 and to the growth control tube.
10. Shake the rack gently to mix the tube contents and place the tubes in the
incubator for 18 to 24 hours.
11. All sets were read visually and MIC values were recorded as the lowest
concentration of the chlorine and chlorine dioxide treatments that had no
visible turbidity depending on positive control and negative control test tubes.
12. MBC (Minimum bactericidal Concentration) was determined by transferring
0.1ml of MIC test tubes and spread on Mueller-Hinton agar. After incubation
time MBC was recorded for each of samples (sewage and artificially
contaminated water) as a lowest concentration of chlorine and chlorine dioxide
that had bactericidal activity if growth not obtained on the agar plate (MIC is
MBC), however if bacterial growth noticed on the agar plate it means that the
MIC is not MBC and the treatment was with bacteriostatic activity.
129.
130.
131.
132. 2) The effect of recommended concentration of Chlorine and
Chlorine dioxide (XINIX) water disinfectants on the tested
isolates with different contact time:
To evaluate the antibacterial activity for recommended
concentration of Chlorine dioxide (XINIX) and chlorine
disinfectants to the normal and contaminated water, we
prepared two set of artificially contaminated sterilized
chlorine free water by two different bacterial cell density
(CFU/ml) depending on (0.5 McFarland Standard ) for
each tested bacterium (E. coli, Salmonella sp.) then each
of chlorine and Chlorine dioxide (XINIX) product were
added separately to different contaminated water
according to recommended concentration in the standard
sheet for both normal and contaminated water. The tested
water (contains: Bacterium inoculums+ recommended
concentration for each of chlorine and XINIX) was
cultivated on Muller Hinton Agar at (5 min) intervals till 1
hour and incubated for 24 hours.
135. Aim
General characters of Enterobacteriaceae
Type of fermentation
Lactose and non- lactose fermenter
Laboratory diagnosis of Escherichia coli
Culture media
Biochemical tests
136. General Characteristics:
Gram negative bacilli, Facultative anaerobic over 40
genera.
Non spore former, some of them non-motile and
other motile by peritrichous flagella.
Habitat (Colon of human and other warm blooded
animals).
Antigenic structure:
All have somatic antigen (O-Ag)
Motile genera have flagellar antigen (H-Ag)
Capsular former like Klebsiella & Salmonella are with (K-Ag and Vi-
Ag) respectively.
Pili antigen (P- and S-Ag).
137. Enterobacteriaceae are characterized by:
Catalase positive, Oxidase negative.
All member of this family are able to reduce
Nitrate(NO3) to Nitrite (NO2)
All are able to degrades sugar (glucose) by
means Embedn Meyerhof pathway and cleave
pyrovic acid to yield formic acid in formic acid
fermentation, end product (Mixed acids end
product and Butanediol or acetoin end product)
from this fermentation are distinguish by
(MR=methyl red , and VP=Voges proskauer).
138. Fermentation pathway
1- The majority carryout (mixed acid fermentation) (Acetic
acid, lactic acid, succinic acid, formic acid and ethanol)
distinguish by (Methyl red test (+ve) result) :
Escherichia coli
2- (Butanediol fermntation) the major product are
(Butanediol, ethanol and CO2) distinguish by (Voges
proskauer test +ve result):
Enterobacter sp.
Serratia sp.
Erwinia sp.
Klebsiella sp.
Citrobacter sp
139. Lactose fermentation useful for distinguish more
pathogenic from less pathogenic or non pathogenic
genera:
DistinctionofPotentialEntericpathogensbylactose fermentation
Lactosefermenter(+ve) Lactosenon-fermenter(-ve)
E.coli Shigella Non-motile
NoH2SproducerKlebsiella Yersinia
Enterobacter
Citrobacter Proteus Motile
H2SproducerSerratia Salmonella
140. Culture media for Enterobacteriaceae:
1- MacConkey agar & Deoxycholate citrate bile salt agar:
Both are (Selective and Differential) How? Selective:
inhibit all Gram-positive bacteria, and differential between
lactose fomenter (appears with pink colonies color) and
Non-lactose fomenter (appears with colorless colonies)
2- Eosin Methylene blue (EMB):
E. coli appears green metallic sheen while other pathogenic genera
are colorless colonies.
3- S.S. agar: (Salmonella-Shigella agar):
Salmonella appears as colorless colonies with black center while
Shigella with colorless colonies without black center. Others
lactose fermenter genera are with pink colonies color.
142. Some important medically important genera in the family
Enterobacteriaceae especially those which cause water
borne diseases are:
a. Lactose fermenter Enterobacteriaceae:
1. Escherichia coli:
Five strains categories of pathogenic E. coli are recognized:
Enterotoxigenic (ETEC).
Enteroinvasive or "Shigella-like" (EIEC)
Enteropathogenic (EPEC).
Enterohemorrhagic (EHEC).
Eteroaggregative (EAEC).
Laboratory diagnosis:
Colonial morphology: lactose fermenter, Small pink colonies on
MacConkey agar, S.S. agar, Deoxycholate citrate bile salt agar while
colorless on Hektoen enteric agar, EMB (eosin-methylene blue appears
with green metallic sheen.
IMViC test and Kligler iron agar KIA
143. Lactose fermenter Non – Lactose fermenter
on Hektoen Enteric agar
Yellow –orange color Green colony
151. Purpose of Lab.
General info. Of V. cholerae
Laboratory diagnosis
Enrichment media
Selective media
Biochemical test
Serological test
152. General characters
Gram-negative, facultative anaerobe, straight or curved
rods bacilli, motile by means of a single polar flagellum
(Monotrichous), with 2 chromosomes.
Vibrio are typically marine organisms; most species require
2-3% NaCl or a sea water base for optimal growth.
Vibrio are one of the most common organisms in surface
waters of the world. They occur in both marine and
freshwater habitats and in associations with aquatic
animals.
Most species are Oxidase-positive
Can ferments glucose, sucrose, and mannitol
153. General characters
Modes of transmissions (fecal contamination of waters,
food for human & animal like shellfish and shrimp).
Infectious does:
Water (infectious dose = 109),
Food (infectious dose = 103)
Person-to-person
Most frequent causative cholera: El Tor biotype of O1 V.
cholera serotype Ogawa.
Divided into two types according to (O-Ag) in the cell wall.
Non O1- group: cause sporadic disease or non pathogen.
O1-group: cause epidemic disease, and with two biotypes
(Based on biochemical reaction):
154. Pathogenic species disease
1. V. cholera Cholera
2. V. parahaemolyticus (salt
friend)
Diarrhea associated with eating raw or improperly
cocked seafood
3. V. vulnivicus (Halophile) Cellulites especially in shellfish handlers
Pathogenic Vibrio
156. Holding or transport media
.1Venkataraman - ramakrishnan (VR) medium:
20g Sea Salt Powde
5g Peptone dissolved in 1L of distilled water.
2. Cary-Blair medium: This most widely-used carrying media. This is a buffered solution
of sodium chloride, sodium thioglycollate, disodium phosphate and calcium chloride
at pH 8.4.
3. Autoclaved sea wate
Enrichment media
1. Alkaline peptone water at pH 8.6
2. Monsur's taurocholate tellurite peptone water at pH 9.2
In epidemic area: Clinical judgment enough.
In pandemic area: detection require the following media and methods :
157. 1- Using Culture media:
A- Non-selective
.1Alkaline bile salt agar (BSA): The colonies are very similar to those on nutrient
agar.
.2Alkaline meat Extract Agar (MEA) smooth, opaque, and cream colored.
B- Selective media
1. MacConkey agar: Colorless colonies because non- lactose fermenter.
2. Hektoen Entric agar : green color appearance.
3. Monsur's gelatin Tauro cholate trypticase tellurite agar (GTTA) medium: Cholera
vibrios produce small translucent colonies with a greyish black centre with halo.
citrate,thiosulphate,containsmediumThis.mediumusedwidelymostlyThis:TCBS.4
bile salts and sucrose. Cholera vibrios produce flat 2-3 mm in diameter, yellow
nucleated colonies.
160. 2. Direct microscopy
. Microscopy is preferred only after enrichment, as this process reveals the
characteristic motility of Vibrios and its inhibition by appropriate antiserum.
3. Biochemical test:
A- Oxidase
B- T.S.I. (Triple Sugar Iron) gar: (slant A/ A Butt) without gas and H2S.
C- Citrate, Ornithine, Mannitol fermentation positive.
161. 4. Serological methods:
a. Agglutination test: Diagnosis can be confirmed as well
as serotype was done by agglutination with specific sera.
Agglutination of bacterium by polyvalent O1 and non O1-
antisera.
b. Titer: By detecting a rise of antibody titer in acute and
convalescent phase sera.
