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Faecal Bacteria Objective To know the types of faecal bacteria prevalent in the aquatic environment and their relevance to Environmental Engineering To know the methods used to enumerate faecal bacteria References Kiely - Environmental Engineering James A & Evison L - Biological Indicators of Water Quality Lecture Outline Faecal Bacteria Methods of Enumeration Faecal Bacteria Non-Pathogenic Escherichia coli * * indicator organisms Streptococcus faecalis* Lactobacillus sp. Enterococcus faecalis, etc Pathogenic TyphoidSalmonella typhi Paratyphoid Salmonella paratyphi Cholera Vibrio cholera Dysentery Shigella dysenteriae Weils Disease Leptospira interrogans (Leptospirosis) (Protozoal Giardiasis; Amoebic Dysentery; Cryptosporidiosis. ViralPolio, Hepatitis A, Gastro enteritis, Aseptic Meningitis, etc.) Problems in Counting Pathogens Techniques Complicated Tissue Culture (Viruses) Cell Enrichment (Bacteria) Techniques Protracted Viruses 2+ weeks Bacteria 1 week Better to count Indicator Organisms Present in faeces always. Indicate the possible presence of a Pathogen Properties of an ‘Ideal’ Indicator Bacteria Should be: Present in high numbers. Specific to faecal material. Identified by simple consistent tests. non-pathogenic. Behave in a similar way to pathogens in the environment. Survival rate same or better than pathogens. As resistant or more resistant than pathogens to disinfection. Bacterial Indicators in Common Use (1) Total Coliforms (TC) Escherichia, Citrobacter, Klebsiella, Enterobacter. Gram negative rods, ferment lactose to acid + gas at 37C Bile (detergent) tolerant - basis of selective media Not always restricted to Faeces Further identification by IMViC Tests Indole production, Methyl Red test, Voges-Proskauer test, Citrate utilization, plus growth at 44.5 C Bacterial Indicators in Common Use (2) Thermotolerant Coliforms (TTC) or Faecal Coliforms (FC) as for Total Coliforms but can grow and ferment lactose at 44.5 C mainly Escherichia coli but includes Citrobacter, Klebsiella, Enterobacter Escherichia coli (E. coli) as above but can also produce Indole from Tryptophan at 44.5 C Always restricted to Faeces Bacterial Indicators in Common Use (3) Faecal Streptococci (FS) Confirm conflicting results from (1) and (2) Better survival then E.coli in cold waters Greater resistance to chlorine Better survival at sea. Distinguish between Animal and Human pollution. Man Sheep Cow Pig FC/FS Ratio > 4.4 0.4 0.2 0.04 Caution only valid for fresh polluion < 24 h. (differential die-off) APHA now recommends use of Streptococcus bovis (animals) and Enterococcus faecalis (man) Bacterial Indicators in Common Use (4) Clostridium perfringens Resistant spores - long survival in water and sediments. – Use to detect remote pollution when few samples taken. e.g. farm supplies, wells, springs. Survives chlorination – Use to check Chlorination efficiency. Survives seawater very well. – Use to check sewage contamination of sea bed. Organisms Present in Raw Sewage Harmless Bacteria E. coli, Coliforms Faecal Streptococci 105 - 109 /100ml Pathogenic Bacteria Salmonella typhi Vibrio cholera Shigella 103 - 104 /100ml Protozoal Entamoeba hystolytica Viral Polio, Coxsackie, Adenovirus 105 - 109 PFU/l Helminths Schistosoma, Ascaris, Taena 5 - 80% of population 102 /l Legionnaires’ Disease (Legionellosis) Legionella pneumophila fever, headache, respiratory symptoms, pneumonia Opportunist pathogens aquatic and terrestrial habitats Water systems cooling towers, spa baths, fountains, distribution mains, air conditioning 20 C minimum stagnation Aerosol formation Risk Assessment Monitoring – Heterotrophic Plate Count – Immunological probes (confirmation) Prevention – eliminate growth conditions – DISINFECTION Enumeration Why enumerate bacteria Quality – Abstraction (75/440/EEC; 79/869/EEC) – Bathing (76/160/EEC) – Drinking (80/778/EEC) Risk Assessment – Disease prevention – Ingestion by Faecal-Oral Route – Aerosols Enumeration How to Enumerate Bacteria Counting by Microscopy – Specific Stains – Time required Culture Techniques – Plate Counts – Selective Agar – Multiple Tube Method – Most Probable Number (MPN) – Membrane Filtration Enumeration Sample Preparation Collection Transport and Storage (6 h max, cool) Aseptic Technique Dilution to Extinction Interpretation of Results sources of error – moribund and stressed cells – clumping and dispersion – experimental Means – arithmetic (normal distribution) – geometric (skewed distribution)