Microbiology

Water
Water

 Man can survive longer

without food but
NOT WITHOUT WATER

 Man requires clean, potable and

safe water
Sources of
Drinking Water

natural sources
stream or water falls

ground water from

deep-wells
Sources of
Drinking Water

Treated piped
water

Bottled water
Sources of Water
REFILLING STATIONS
 Water
 sometimes these sources may be
contaminated with:
– chemicals
– radioactive materials
– microorganisms

– at a dangerous level or concentration

 Water Microbiology
What is microbiology/microorganism?
How do microorganisms look like?
What do they require for growth?
Where do they come from?
What microbes are found in water?
 Water Microbiology
Are these organisms harmful?
If so, what are the diseases they cause?
What are the indicators that water is
contaminated / polluted?
How are these organisms detected?
Bacteriological Tests
What water samples are tested?
How are the samples collected?
How are the samples transported?
How are the samples processed?
How are the results interpreted?
What are the standards for safe
drinking water?
Microbiology
 The study of
microorganisms
– Very small
• microscopic

• also called microbes or

germs
 protozoa
Microorganisms virus

bacteria
bacteria

bacteria

virusbacteria
bacteria
virus
 Microbiology
 What
are
BACTERIA?
– Bacterium
(singular)
– Prokaryotic
– unicellular
(one-celled)
Microbiology
 What are FUNGI?
– fungus (singular)
– eukaryotic
– non-
photosynthetic
microbes
– saprophytic or
parasitic
Microbiology
 What are
PROTOZOA?
– eukaryotic microbes
– frequently found in
water and soil
– some are pathogens
– some motile
– usually unicellular
Microbiology
H
 What are VIRUSES? O
– virus (singular) S
T
– acellular C
– smaller than bacteria E
– obligate intracellular L
L
parasites of
 Plants
 Animals
 Humans
 bacteria
 Microbiology
 What are ALGAE?
– Alga (algae)
– Eukaryotic
– photosynthetic organisms
– range in size from
unicellular to multicellular
– includes many seaweeds
 Growth Requirements
 Nutrients

– Macronutrients:

– Carbon, hydrogen
– oxygen, nitrogen
– phosphorus, sulfur
 Growth Requirements
 Nutrients
– Micronutrients:
– Sodium, potassium, chlorine
– magnesium, calcium, iron
– iodine, other trace elements

– carbohydrates, fats, proteins, nucleic acids

 Microbial Growth
 Other requirements

pH

temperature
Microbial Growth – other reqts
 WATER IS A MEDIUM
FOR GROWTH AND
REPRODUCTION
OF MICROORGANISMS !
Microbes come from

plants

air soil

people animals water

Microbes in humans / animals
 Indigenous microflora
(normal flora)
– skin
– mouth
– ear
– eye
Microbes in humans / animals
 Indigenous microflora
(normal flora)
– urogenital area
– gastrointestinal tract
Microbes in humans / animals
 Pathogens
– microorganism that
cause diseases
– carriers –
 harbor the organism
 but NO disease
MICROORGANISMS ARE
FOUND EVERYWHERE,
ON US, IN US AND
AROUND US !
Sources of Water
Contamination
Air & water
Sewage
human & animal wastes

improper handling and storage

Soil and Air Microbes

 Pseudomonas
 Flavobacterium
 Acinetobacter
 Klebsiella
 Serratia
 Soil and Air Microbes
 when in high numbers

 may cause opportunistic infections in

– immunosuppressed persons
– very young
– old persons
– pregnant
 Sewage

 Waste and wastewater

 discharged into sewers
 by residential and commercial
establishments
 Sewage

 may contain organic & inorganic matter,

(detergents, pharmaceuticals, petroleum-
based oil, heavy metals and pesticides )

 it also contains bacteria, viruses,

protozoa, many of which are pathogenic
 Water Borne Diseases
 caused by organisms found in human and
animal wastes (fecal contamination)
 In the feces, there are:
– normal bacterial flora of the intestines
– pathogens

 contamination with feces

 Normal Bacterial Flora

 causesof opportunistic and hospital

acquired infections in
immunocompromised individuals
 Pathogenic Organisms
 true pathogenic organisms
– Salmonella
– Shigella
– enterotoxigenic Escherichia coli
– Vibrio cholerae
– Yersinia enterocolitica
– Campylobacter fetus
 Pathogenic Organisms
 may cause clinical syndromes :
 mild gastroenteritis
– symptoms: vomiting & diarrhea, fever
– prolonged vomiting & diarrhea may lead to
dehydration then death
 Pathogenic Organisms
 may cause clinical syndromes :
 serious disease such as typhoid fever
– organism is in the blood may affect other
organs of the body
 Water Borne Diseases
 Cholera  Dysentery
 Botulism  Cryptosporidiosis
 Typhoid  Polio
 Hepatitis A  Giardia
 Water Borne Diseases
 Routeof
transmission -

–gastrointestinal

–drinking of
contaminated water
 Water Borne Diseases
Route of transmission -

 Skin contact
Water Borne Diseases

 Bacteria : Cholera, Botulism, Typhoid

 Vibrio cholerae
 Clostridium botulinum (can be thru food)
 Salmonella typhi (can be thru food)
Water Borne Diseases

 Virus : Hepatitis A, Polio

 Hepatitis A virus (also thru food)
 Polioviruses (exposure to untreated
sewage)
 Water Borne Diseases
 Protozoa : Cryptosporidiosis, Giardiasis
 Cryptosporidium parvum (resists
chlorine)
 Giardia lamblia
 Water Borne Diseases
 Dysentery: Bacteria and Protozoa
– diarrhea with blood and mucus
 Shigella dysenteriae bacteria
 Entamoeba histolytica
 both also thru food
 Determination of
contamination
 pathogens are excreted in the
human and animal feces
 may contaminate water source
 laboratory can isolate the
pathogens from water
 Determination of
contamination
 the laboratory methods:
– isolation and enumeration
– complicated and time-consuming
 impractical
– to monitor for every possible
pathogen
 Determination of
contamination
 easier
to detect organisms that are
normally present in the feces

 anaerobes
 COLIFORMS - organisms present
in the intestinal tract (colon)
 Determination of
contamination
 COLIFORMS as indicators


– of fecal pollution
– for the efficacy of water treatment
and disinfection
 Indicator Organisms
 Escherichia coli
 Thermotolerant coliform bacteria
 Coliform organisms (total coliforms)
 Fecal streptococci
 Non-fecal
Fecal

Total coliform

Fecal coliform

E. coli
 Coliform organisms
 Any rod-shaped, non-spore-
forming, Gram-negative
bacteria
capable of growth in the
presence of bile salts (or other
surface-active agents)
Coliform organisms
 cytochrome-oxidase
negative
 ferment lactose at
either 35 or 37°C
produce acid, gas
 aldehyde within 24-48 h
Coliform organisms
 coliform group includes species from
the genera :
 Escherichia
 Klebsiella

 Enterobacter

 Citrobacter

–includes Escherichia coli

 HPC
 Detection of coliforms to document
fecal pollution is NOT enough to make
a conclusion that the water is not
potable or safe for drinking

 Heterotrophic plate count (HPC)

 HPC
 Presence of these organisms
reflects :
– the general cleanliness of the water
distribution system
– Quality of the bottled water
 HPC
 to estimate the number of live
heterotrophic bacteria
– utilizes preformed organic materials from
environment as source of food
 can not synthesize from atmospheric
carbon dioxide
 HPC
 other organisms, not necessarily coming
from human and fecal wastes may
contaminate water
 a high HPC may mask
the presence of coliforms
 Water Sampling
 Representative of the whole water
supply system:
– water source
– before & after treatment (if any)
 from a reasonable number of points in
the distribution system
 Water Samples
 Level I (Point source)
 Level II (Communal Faucet System or
standposts)
 Level III (Waterworks System or Individual
House Connections)
 Bottled drinking water
 Emergency supply of drinking water
 Bottles
 Sterile / autoclaved
  100 mL
 with space for shaking
of specimen
 Covered with foil or x
paper (sterile)
 Water Samples

Level I (Point source)

– a protected well or a developed spring
– with outlet but NO distribution system,
 Water Samples
Level II
(Communal Faucet Sx or Standposts)
 a system with a source, a reservoir, a
piped distribution network & communal
faucets
 Water Samples
Level III (Waterworks System or
Individual House Connections)
 a system with a source, a reservoir, a
piped distribution network & household
taps
 urban areas
 Water Samples
Bottled Water
 water that is placed in a sealed container
or package
 offered for sale for human consumption
as drinking water
 Frequency of Sampling
 Based on the mode & source of water
supply
– Bottled water once every 2 months
 past frequency of records yielding
unsatisfactory results
 the quality of raw water treated
 number of raw water sources
 Frequency of Sampling
 the adequacy of treatment
 capacity of the treatment plant
 risks of contamination at the source & in
the distribution system
 risk of an epidemic
 practice of disinfection
 Frequency of Sampling
Source / Mode Min Frequency Population
of Supply

 Level I once every 3 months (90-150)

 Level II once every 2 months (600)

 Level III 1 sample monthly (<5,000)

 Frequency of Sampling
Source / Mode Min Frequency Population
of Supply

 Level III 1 sample per 5 K pop

monthly (5 to 100 K)

20 samples plus 1 sample

per 10 K pop monthly (>100 K)
 Frequency of Sampling
Source/Mode of Supply Min Frequency

 Bottled drinking water once every 2 months

 Refilling station once a month

 Emergency supplies of before delivery to

drinking water users
Methods of Sampling

 Should be representative

 Avoid contamination during:

– collection &
– before examination
 Methods of Collection
LEVEL I
 pump water to waste for 2 min
 Flame mouth of pump (optional)
 sample direct from mouth of pump
 Methods of Collection
LEVEL II
 allow water to waste for  1 min
 Flame tap (optional)
 samples taken from:
– farthest standpipe
– entry point of the distribution system
 Methods of Collection
LEVEL III
 remove any filters
 allow water to waste for  1 min
 Flame tap (optional)
 samples taken from:
– source of the distribution system
– taps at strategic sampling points
 Methods of Collection
EMERGENCY SUPPLY OF DRINKING WATER
 sample is taken :
– before being served to end-users
 sample is taken at :
– storage containers
– mobile tankers
 Methods of Collection
BOTTLED DRINKING WATER
 as water taken from source
 sample is taken from manufacturer’s
stock & outlets
 Transport of samples
 Transport < 2h NO ICE
 Time between sample collection and
analysis:
– NOT more than 6 h
– 24 h absolute MAXIMUM
 ice box (styro, ice chest, water cooler)
 add ice and water
 Transport of samples
 If delay is unavoidable
– filter water and place in medium

 If NOT: sample is REJECTED

 Transport of samples
Storage / Transport condition:
 temperature
 state of container and sample are
NOTED
 used in interpretation of results
 Transport of samples
 if sample contains chlorine (even in
trace amount)

 bottle must contain sodium thiosulfate

to neutralize chlorine
 Transport of samples

 Box cleaned and disinfected after use

 to avoid contamination
– bottles
– sampler’s hands
 Analytical Techniques
 Membrane-filtration method
 Multiple-tube method or Most Probable
Method (MPN)
 Presence-absence tests
 Analytical Techniques
Other methods (alternative)
 PHC Test
 MMONPG-MUG
Membrane-filtration method
 Membrane filter 0.2
to 0.45 m

 vacuum pump
 Membrane-filtration method
 Petri dish with
medium
 Organisms on top
and within filter
 will grow and
counted
 Membrane-filtration method
 For processed
water, low turbidity

 Disadvantages:
 expensive filter
 special equipment
 Multiple tube or MPN
 Estimate of the microbes in the original
sample is made (statistical)
 Highly turbid samples and for drinking-
water analysis
 Presumptive, Confirmed and Completed
tests
Water Presumptive

Confirmed

Completed
 Multiple tube or MPN
 Analysis of a series of volumes of
sample
 3- or 5-tube method
– volumes of 10, 1 and 0.1 ml
– if contaminated water, extended to
0.01, 0.001 ml
 Multiple tube or MPN
 water is added to liquid medium
(Lactose broth or Lauryl tryptose broth)
with inverted (Durham) tube
– Turbidity shows growth
– lactose fermentation with gas production
– observed at 24 and 48 h
 Presumptive

inoculate

24 to 48h 35C

Negative Doubtful
Presumptive Result Positive
 Confirmed

Negative Doubtful
Presumptive Result Positive

Streak
EMB
24 to 48h 35C
 Completed
Dark-centered

Negative
Coliform
Greenish GNB
metallic NSF
sheen Lac +
24 at 35C

Lactose broth NA slant

 MPN is 15
coliforms per
100 ml of
sample
collected
 Presence-absence tests
 Monitoring good-quality drinking water,
POSITIVE results are RARE
 qualitative not quantitative
 indicate only the presence or absence
of the indicator organism tested
 Analytical Techniques
PHC Test
 simple, inexpensive & rapid
 screening test
 detection of coliform organisms
 NOT substitute for standard water tests
Analytical Techniques
MMONPG-MUG
 Minimal medium ONPG
(orthonitrophenol--D-
galactopyranoside) and
MUG (4-methylumbelliferyl- -
D-glucoronidase)
 Standard Values for
Bacteriological Quality
 DENR Administrative Order No. 26-A
Series 1994
 Philippine National Standards for
Drinking Water 1993 (PNSDW 1993)
 under the Provision of Chap II, Section
9 of PD 856 (Code on Sanitation of the
Philippines)
 PNSDW 1993
Standard Values for Bacteriological
Quality
ALL drinking water Std value
supplies under all
circumstances E. coli 0 per 100 ml
Level I, II, III
Bottled Water Thermotolerant
Emergency Water (fecal coliform)
Supplies 0 per 100 ml
 PNSDW 1993
Microbiological Requirements
 drinking water must be FREE from
pathogens
– bacteria, viruses, protozoans & helminths

 source - free of feces

 treat fecally contaminated water
Thank you !
 Bottled water
 Microbiological specifications at the
SOURCE & at critical control points
 Coliforms: from a set of samples
1 may contain MPN 2.2/100 mL
no sample with MPN 9.2 / 100 mL
 HPC: at various sampling points
not > 300 CFU/ mL
 Bottled water
 Coliforms 1 MPN/100 mL
not E. coli
 Fecal Streptococci 1 cfu/100mL
 Pseudomonas aeruginosa 0 / 100mL
 HPC 1,000,000 cfu/mL
 Purified 100,000 cfu/ml