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Microbiology of Water & Air. Dr.Rouchelle Tellis Assoc Prof, Microbiology. Potable water. Clear, colorless, without dis-agreeable taste & odour Safe: free from harmful toxins & pathogenic Micro-organisms. Lesson plan. Water-borne diseases Pathogen indicators Coliform bacteria
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Microbiology of Water & Air Dr.Rouchelle Tellis Assoc Prof, Microbiology
Potable water • Clear, colorless, without dis-agreeable 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 1991 Cholera Epidemic 1,000,000 cases/10,000 deaths • Bacteria • Virus • Protozoa • Helmiths
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, 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
Water sample Collection • Routine and regular exercise: Appropriate sterile sampling container with Sodium thio-sulphate to inactivate residual chlorine • 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 • Thermo-tolerant Fecal coliforms • Grows at 44.5 C • Escherichia coli • Individual species • Enzyme specific total coliform 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 coliforms 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 MA broth, incubated at 440 C . • Thermo-tolerant 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
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 Chlorine Demand Kill Free Residual Chlorine Combined Residual Chlorine Organic
Bacteriology of Air • Air borne infections: 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
Viral infections that spread through air: • 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 pate and mechanically rotated