Faecal Bacteria v Objective w To know the

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Faecal Bacteria v Objective w To know the types of faecal bacteria prevalent in

Faecal Bacteria v Objective w To know the types of faecal bacteria prevalent in the aquatic environment and their relevance to Environmental Engineering w To know the methods used to enumerate faecal bacteria v References w Kiely - Environmental Engineering w James A & Evison L - Biological Indicators of Water Quality v Lecture Outline w Faecal Bacteria w Methods of Enumeration

Faecal Bacteria v Non-Pathogenic w w Escherichia coli * * indicator organisms Streptococcus faecalis*

Faecal Bacteria v Non-Pathogenic w w Escherichia coli * * indicator organisms Streptococcus faecalis* Lactobacillus sp. Enterococcus faecalis, etc v Pathogenic w w w Typhoid. Salmonella typhi Paratyphoid Salmonella paratyphi Cholera Vibrio cholera Dysentery Shigella dysenteriae Weils Disease Leptospira interrogans (Leptospirosis) (Protozoal Giardiasis; Amoebic Dysentery; Cryptosporidiosis. Viral. Polio, Hepatitis A, Gastro enteritis, Aseptic Meningitis, etc. )

Problems in Counting Pathogens v Techniques Complicated Tissue Culture (Viruses) Cell Enrichment (Bacteria) v

Problems in Counting Pathogens v Techniques Complicated Tissue Culture (Viruses) Cell Enrichment (Bacteria) v Techniques Protracted Viruses 2+ weeks Bacteria 1 week v Better to count Indicator Organisms w Present in faeces always. w Indicate the possible presence of a Pathogen

Properties of an ‘Ideal’ Indicator Bacteria Should be: v Present in high numbers. v

Properties of an ‘Ideal’ Indicator Bacteria Should be: v Present in high numbers. v Specific to faecal material. v Identified by simple consistent tests. v non-pathogenic. v Behave in a similar way to pathogens in the environment. v Survival rate same or better than pathogens. v As resistant or more resistant than pathogens to disinfection.

Bacterial Indicators in Common Use (1) Total Coliforms (TC) w w Escherichia, Citrobacter, Klebsiella,

Bacterial Indicators in Common Use (1) Total Coliforms (TC) w w Escherichia, Citrobacter, Klebsiella, Enterobacter. Gram negative rods, ferment lactose to acid + gas at 37 C Bile (detergent) tolerant - basis of selective media Not always restricted to Faeces Further identification by IMVi. C 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) w

Bacterial Indicators in Common Use (2) Thermotolerant Coliforms (TTC) or Faecal Coliforms (FC) w as for Total Coliforms but can grow and ferment lactose at 44. 5 C w mainly Escherichia coli but includes Citrobacter, Klebsiella, Enterobacter Escherichia coli (E. coli) w as above but can also produce Indole from Tryptophan at 44. 5 C w Always restricted to Faeces

Bacterial Indicators in Common Use (3) w w w Faecal Streptococci (FS) Confirm conflicting

Bacterial Indicators in Common Use (3) w w w 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. 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 w Resistant spores - long survival

Bacterial Indicators in Common Use (4) Clostridium perfringens w Resistant spores - long survival in water and sediments. – Use to detect remote pollution when few samples taken. e. g. farm supplies, wells, springs. w Survives chlorination – Use to check Chlorination efficiency. w Survives seawater very well. – Use to check sewage contamination of sea bed.

Organisms Present in Raw Sewage v Harmless Bacteria w E. coli, Coliforms w Faecal

Organisms Present in Raw Sewage v Harmless Bacteria w E. coli, Coliforms w Faecal Streptococci 105 - 109 /100 ml v Pathogenic Bacteria w Salmonella typhi w Vibrio cholera w Shigella 103 - 104 /100 ml v Protozoal Entamoeba hystolytica v Viral Polio, Coxsackie, Adenovirus 105 - 109 PFU/l v Helminths Schistosoma, Ascaris, Taena 5 - 80% of population 102 /l

Legionnaires’ Disease (Legionellosis) v Legionella pneumophila w fever, headache, respiratory symptoms, pneumonia v Opportunist

Legionnaires’ Disease (Legionellosis) v Legionella pneumophila w fever, headache, respiratory symptoms, pneumonia v Opportunist pathogens w aquatic and terrestrial habitats v Water systems w cooling towers, spa baths, fountains, distribution mains, air conditioning w 20 C minimum w stagnation v Aerosol formation v Risk Assessment w Monitoring – Heterotrophic Plate Count – Immunological probes (confirmation) w Prevention – eliminate growth conditions – DISINFECTION

Enumeration v Why enumerate bacteria w Quality – Abstraction (75/440/EEC; 79/869/EEC) – Bathing (76/160/EEC)

Enumeration v Why enumerate bacteria w Quality – Abstraction (75/440/EEC; 79/869/EEC) – Bathing (76/160/EEC) – Drinking (80/778/EEC) w Risk Assessment – Disease prevention – Ingestion by Faecal-Oral Route – Aerosols

Enumeration v How to Enumerate Bacteria w Counting by Microscopy – Specific Stains –

Enumeration v How to Enumerate Bacteria w Counting by Microscopy – Specific Stains – Time required w Culture Techniques – Plate Counts – Selective Agar – Multiple Tube Method – Most Probable Number (MPN) – Membrane Filtration

Enumeration v Sample Preparation w w Collection Transport and Storage (6 h max, cool)

Enumeration v Sample Preparation w w Collection Transport and Storage (6 h max, cool) Aseptic Technique Dilution to Extinction v Interpretation of Results w sources of error – moribund and stressed cells – clumping and dispersion – experimental w Means – arithmetic (normal distribution) – geometric (skewed distribution)