Microbiology of Water & Air

1. Microbiology of Water & Air Dr.Rouchelle Tellis Assoc Prof, Microbiology

2. Potable water • Clear, colorless, without dis-agreeable taste & odour • Safe: free from harmful toxins & pathogenic Micro-organisms

3. Lesson plan • Water-borne diseases • Pathogen indicators • Coliform bacteria • Streptococcus • Enterococcus • Enumeration Methods • Membrane filter • Presumptive coliform count • Differential coliform count • Surface Water Standards

4. Water borne pathogens 1991 Cholera Epidemic 1,000,000 cases/10,000 deaths • Bacteria • Virus • Protozoa • Helmiths

5. 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

6. Viral pathogens in water • Enteritis, diarrhea, and dysentery • Rotavirus, Enterovirus • Norwalk • Hepatitis: • Hepatitis A • Hepatitis E • Paralysis • Polio

7. Protozoa • Giardia • Cryptosporidia • Amoeba

8. Helmith • Round worm • Tape worm • Whip worm

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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.

18. 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.

19. 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

20. ONPG-Total Coliform MUG- E. coli

21. Chlorination of Water

22. 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

23. Chlorine Dosage Inorganic Chlorine Demand Kill Free Residual Chlorine Combined Residual Chlorine Organic

24. 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

25. 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

26. Viral infections that spread through air: • Influenza viruses • Rubella virus • Mumps virus • Adenovirus • Parvo-virus B19

27. Mechanism of spread of infection • Droplets and droplet nuclei while coughing, sneezing and talking

28. 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.

29. 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