Download Microbiology of Water

Microbiological Examination
of Water & Air
Potable water
• Clear, colorless, without disagreeable taste &
odour
• Safe: free from harmful toxins & pathogenic
Micro-organisms
Lesson plan
• Water-borne diseases
• Pathogen indicators
– Coliform bacteria
– Streptococcus
– Enterococcus
• Enumeration Methods
– Membrane filter
– Presumptive coliform count
– Differential coliform count
• Surface Water Standards
Water borne pathogens
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Bacteria
Virus
Protozoa
Helmiths
1991 Cholera Epidemic
1,000,000 cases/10,000 deaths
Bacteria pathogens in water
• Enteritis, diarrhea, and dysentery
– Campylobacter
– V. cholera
– E. coli 0157:H
– Salmonella, Shigella
• Enteric fever
– Typhoid, Paratyphoid
• Paralysis: Botulism
• Eye, ear, and skin infections
– P.aeruginosa, M.marinum
Viral pathogens in water
• Enteritis, diarrhea, and dysentery
– Rotavirus, Enterovirus
– Norwalk
• Hepatitis:
– Hepatitis A
– Hepatitis E
• Paralysis
– Polio
Protozoa
• Giardia
• Cryptosporidia
• Amoeba
Helmith
• Round worm
• Tape worm
• Whip worm
Indicator Organisms
• General coliforms – indicate water in contact
with plant or animal life (universally present)
• Fecal coliforms – mammal or bird feces in
water
• Fecal streptococci– feces from warm blooded
animals
• Clostridium perfringens
Sample Collection
• Routine and regular exercise
• Appropriate sterile sampling container with Sodium
thiosulphate to inactivate residual chlorine
• After collection place in a refrigerator
• Deliver to laboratory soon,not later than 6 hours
• Maintain records
Coliform Group (total coliform)
• Enterobacteriaceae
– Facultative anaerobe
– Gram negative
– Non-spore forming
– Rod shaped
– Ferment lactose
– Produce gas and acid
within 48 h @ 35 C
• Coliform genera
– Enterobacter
– Klebsiella
– Citrobacter
– Escherichia
Coliform Group
• Total coliform
• Thermotolerant Fecal
coliform
total coliform
– Grows at 44.5 C
• Escherichia coli
– Individual species
– Enzyme specific
fecal
coliform
E. coli
Streptococcus and Enterococcus
• Fecal Strep
– S. faecalis
– S. faecium
– Resistant to 450C,
40% bile,
potassium tellutite
& Sodium azide
• Fecal Streps that
survive in 6.5%
sodium chloride
Plate count
• No. of colonies formed in nutrient agar pour plate
cultures of water samples are counted
• Incubated aerobically parallel at 370C and 220C
• Plate count at 370C:
indicator of fecal pollution
With potentially pathogenic
bacteria
Membrane Filter Method:
• Filter water through a 0.45 μM membrane
filter
• Place membrane on selective media
• Incubate
– 350 C total coliform
– 44.50 C fecal coliform
• Count the No. of colonies
Presumptive coliform count: Multiple
Tube Fermentation Method
• An estimate of the No. of coliforms is made by adding
varying quantities of water (0.1- 50 ml) to bile salt
lactose peptone water or double strength Mac Conkey
broth.
• Acid and gas formation indicates coliform growth
• Probable No. of coliforms per 100 ml is read from
Most-Probable-Number (MPN) table of McCardy.
Differential coliform count- Eijkman test
•To find out whether the coli forms of bacteria detected in
presumptive test are E.coli.
• After usual presumptive test, subcultures are made from all the
bottles showing acid and gas production to fresh tubes of single
strength MacConkey broth, incubated at 440 C .
•Thermotolerant E. coli give definite proof of fecal pollution.
• Those showing gas in Durham’s tubes, contain E.coli.
•Confirmation of E.coli can be done by testing for indole
production and citrate utilization.
Enzyme Substrate or Chromogenic
Substrate Method
• Total coliforms have the enzyme
– β-D-galactosidase which hydrolyses
– ortho-nitrophenyl- β-D-galactopyranoside (ONPG)
– Yellow when hydrolyzed
• E. coli has the enzyme
– β-glucuronidase which hydrolyses
– 4-methylumbelliferyl-β-glucuronide (MUG)
– Fluoresces when hydrolyzed
ONPG-Total Coliform
MUG- E. coli
Chlorination of Water
Methods of Treatment
• Shock Chlorination (50-100 ppm, contact of at least 6
hours)
• Continuous Chlorination – for recurring bacterial
contamination problems – a measurable amount of free
residual chlorine
Chlorine Dosage
Inorganic
Organic
Chlorine
Demand
Combined
Residual
Chlorine
Free
Residual
Chlorine
Kill
Bacteriology of Air
• Air borne infection: transmission of infection
produced by respiratory droplets less than 5um
in size
• Droplet infection: transmission of infection
produced by respiratory droplets larger than
5um in size
• Pathogenic bacteria do not multiply in air
Pathogenic organisms spread
through air
Bacterial:
– Streptococcus pyogenes
– M.tuberculosis
– N.meningitidis
– C.diphtheriae
– H.influenzae type B
– B.pertussis
– Y.pestis (pneumonic plague)
– Mycoplasma pneumoniae
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Viral infections that spread through
air:
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Influenza viruses
Rubella virus
Mumps virus
Adenovirus
Parvo-virus B19
Mechanism of spread of infection
• Droplets and droplet nuclei while coughing,
sneezing and talking
Measurement of air contamination
Sedimentation ‘Settle Plate method’
• Estimating the No. of bacteria in air by permitting
bacteria to settle on open perti dishes containing
culture media over a fixed duration.
Slit sampler
• A means of estimating the No. of bacteria present
in the air by passing a known volume of air
through the ‘slit’ 0.25mm wide.
• Air is directed onto a
• Plate and mechanically
• rotated