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

1991 Cholera Epidemic 1,000,000 cases/10,000 deaths 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

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

Kill Inorganic Free Residual Chlorine Organic Chlorine Dosage Combined 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