Pathogens

• Disease causing microorganisms, such as

bacteria, fungi, and viruses, found commonly
in sewage, hospital waste, run-off water from
farms, and in water used for swimming.
• Most pathogens are parasites and the
diseases they cause are an indirect result of
their obtaining food from, or shelter in, the
host.
 Groups of microbes present in water
• Fluorescent bacteria (Pseudomonas,
alginomonas)
• Chromgenic rods (Xanthomonas)
• Coliform group (E.coli, Aerobacter)
• Proteus group
• Spore formers of genus (Bacillus)
• Pigmented and non-pigmented coci
(Micrococcus)
Aquatic fungi in different water bodies
Aquatic fungi Source
Achlya americana Tap water, fish pathogen

A. Laevis Lake water, fish pathogen

Pythium undulatum Pond water, fish pathogen

Dictyachus psci Pond water, fish pathogen

 Water-borne pathogenic bacteria
Bacteria Reservoir Other features
Aeromonas hydrophila Free-living gastroenteritis

Chromobacterium sp Soil run off saprophyte

Mycobacterium tuberculosis Free-living, infected tuberculosis

person
Vibrio cholerae Free-living Causes diarrhoa

Salmonella enteritditis Intestine of animals Water-borne

Yersinia enterocolitica In animals Water-borne,

gastroenteritis

Pseudomonas aeruginosa Free-living Swimer’s ear

 Water-borne pathogenic protozoa
Protozoa reservoir Other features

Giardia lamblia Sheep, dog, cat Diarrhoea

Cryptosporidium On domestic and wild Acute enterocolitis

animals
Acanthmoeba Sewage sludge disposal Amoebic encephalitis
area

Entamoeba histolytica water Amoebiasis

Ways by which microorganisms enter
in water supply
 Broken sewer lines
 Inappropriate treatment
 Unhygienic environment at public places of
water collection
 People suffering from communicable diseases
also discharge water through their excreta
(amoebic dysentery, typhoid fever, bacillary
dysentery, poliomyelitis etc., )
 Coliforms
• The characteristic group of intestinal bacteria
are the coli forms
• They are defined as facultative anaerobic,
Gram-negative, nonspore-forming rod shaped
bacteria that ferment lactose with gas and
acid formation within 48 hours at 350C.
 Coliforms
• Many facultative anaerobic, Gram-negative,
non-spore-forming, rod-shaped bacteria that
develop red colonies with a metallic (golden)
sheen within 24 hours at 37°C on an Endo-
type medium containing lactose.
 Coliforms
• All bacteria possessing the enzyme β-
galactosidase, which cleaves a chromogenic
substrate (e.g., ortho-nitrophenyl-β-D-
galactopyranoside), resulting in release of a
chromogen (ortho-nitrophenol).
 Indicator organisms
• The coli form group make up about 10% of
intestinal microorganisms of human and
animals
• Thy are used as indicator organisms, as an
index of possible water contamination
• If coli forms are not detectable in 100ml, the
water can be said to be potable water
 Selected coliform bacteria in the family
Enterobacteriaceae
b
ortho-Nitrophenyl-β-D-galactopyranoside.

ONPGb Faecal origin

Non faecal
origin

Budvicia + - +
Citrobacter + + +
Enterobacter + + +
Erwinia + - +
Escherichia + + -
Klebsiella + + +
Leclercia + - +
Serratia + - +
y tests for coliforms
Differentiation of E.coli and A. aerogenes on eosin or EMB agar
Thomas formula
observation
• Calculation Case (i)
For three each of 10 mL, 1 mL and 0.1 mL sample
concentration combinations
MPN from the MPN table = .........
• Case (ii)
For other combinations and dilutions using
Thomas formula

Result
MPN/100 mL = .........
 MPN Table
Number of positive lactose broth tubes Number of coli
form organisms
10ml 1ml 0.1ml
0 0 0 0
0 1 0 2
0 1 1 4
1 0 0 2.2
1 0 1 4
1 1 0 4.4
2 0 0 5
2 0 1 7
2 1 0 7.6
3 0 0 8.8
3 0 1 11
3 1 0 12
 MPN Table
Number of positive lactose broth tubes Number of coli
form organisms
10ml 1ml 0.1ml
4 0 0 15
4 0 1 20
4 1 0 21
5 0 0 38
5 0 1 96
5 1 0 240
The Membrane filter technique
• The filtering apparatus consists of a glass or
stainless steel funnel and a flask
• The funnel is clamped to a base containing a
filter
• The stem of base is inserted into a filter flask
through a rubber stopper
• A sterile membrane filter (0.45µm) disk is placed in the
sterilized holding apparatus
• A volume of water is passed through filter disk
• Bacteria present in water sample are retained on filter disk
• The side of funnel and membrane are rinsed with sterile
distilled water
• The membrane filter disk is aseptically removed and placed
in a petridish with medium
• The medium passes through the pores of membrane and
nourishes the bacteria present on it
• After incubation at 350C for 24 h the colonies are counted
Calculation
 Advantages
• It permits small numbers of bacteria from
large volume of water. So more accurate and
reliable
• It allows direct counting of bacteria
• Time saving method
 Disadvantages
• In turbid water containing algal growth and
other materials the pores are clogged
• High populations of non-coliforms and other
bacteria cause overgrowth
• Metals and phenols can be absorbed to
membrane filter and therefore inhibit growth
of bacteria retained on membrane filter
Faecal streptococci
• Streptococci are present in human and animal
intestines.
• Many species of streptococcus are pathogenic.
• They cause diseases such as bacterial pneumonia, ear
infection and bacterial meningitis.
• Faecal streptococci (enterococcus) are a subgroup of
the genus streptococcus.
• Like faecal coliforms, faecal streptococci are applied as
indicators for water pollution.
• The presence of faecal streptococci indicates the
presence of faecal pathogens in water.
Clostridium as an indicator organisms
Clostridium perfringens has one
of the highest reproduction
rates in the bacteria kingdom.
Under optimal conditions, the
cell doubles every 10 minutes!
• Only Clostridium perfringens is specific to faecal material,
with other species being found in soil and other
environmental materials.
• Clostridia can form spores that are extremely resistant to
the water environment and water treatment processes.
• As a consequence, their presence in the absence of E. coli
and enterococci can be taken to indicate a historic pollution
event.
• Cl. perfringens is present in lower numbers than E. coli or
enterococci in the faeces of all animals
• The spores are resistant to a wide range of environments.
 Detection of Clostridium
perferingens
• Two-step membrane-filtration technique
• The sample is filtered through a 0.45µm membrane and the filter is
transferred to an mCP agar plate.
• The plates are incubated anaerobically (either in an anaerobic jar or
bag) at 44.5°C for 24 hours.
• The mCP agar contains differential and selective agents. D-
cycloserine and polymyxin B sulfate are antibiotics that inhibit
background growth.
• Bromocresol purple identifies colonies, including C. perfringens that
can ferment sucrose.
• Phenolphthalein diphosphate identifies colonies that produce acid
phosphatase, such as C. perfringens.
• After incubation on mCP, filter membranes containing
yellow straw-colored colonies are transferred to pads
saturated with ammonium hydroxide in a chemical
fume hood.
• Following a 15 second exposure, colonies that turn
dark pink to magenta are counted as C. perfringens.
• The color change upon exposure to ammonium
hydroxide is an indication that acid phosphatase has
cleaved the phosphate in phenolphthalein
diphosphate.
 Membrane Clostridium perferingens medium
(A medium for rapid isolation and presumptive identification of
Clostridium perfringens from water samples).
Typical Formula* gm/litre
Tryptose 30.0
MEMBRANE CLOSTRIDIUM PERFRINGENS (m-CP) MEDIUM
Yeast Extract 20.0
medium for rapid isolation and presumptive identification of
Sucrose 5.0
Clostridium perfringens from water samples.
L-cysteine hydrochloride 1.0
Magnesium
0.1
sulphate.7H2O
Agar 15.0
Bromocresol purple 0.04
pH 7.6 ± 0.2 @ 25°C
 Typical colony on M-cp agar
Organism Typical Colony Colour
Opaque Yellow
Clostridium perfringens Sucrose positive/Glucosidase negative
then pink/red after exposure to NH4OH
Blue/Green
Sucrose positve/Glucosidase positive(e.g.
Cl. baratii, Cl. paraputrificum, Cl. tertium)
Purple
Sucrose negative/Glucosidase positive or
Other Clostridium spp.
negative (e.g. Cl. biferentans, Cl. difficle,
Cl. sporogenes)
Opaque Yellow
Sucrose positive/Glucosidase negative
remain yellow after exposure to NH4OH