Water microbiology

Water Bacteriology

Water Microbiology

• Study of microorganisms and their communities in water environment is called Aquatic microbiology, • while Water Microbiology relates to the study of

microorganisms in potable water.

• The scope of Aquatic Microbiology is wide and includes the habitats like planktons, benthos, microbial mats and biofilm which may be found in lakes, rivers, streams, seas, groundwater, rain, snow and hail.

The Essentialness of Water Blood in our veins

approximates composition of sea water

Concept of hydrophilic and hydrophobic nature of biological molecules

These molecules determine shape of biological molecules and thus decide the specificity of all living processes

Essential for All living organisms

Water covers 70% of the world

97% of the water is in the oceans

• Drinking or potable water is water that is free from pathogens and chemicals that are dangerous to human health.

• Any taste, odor and color must be absent from the water to be palatable.

• Raw water may contain many contaminants derived from sewage and nearby industries.

• Many enteric pathogens are water borne.

• Therefore water is treated and disinfected to remove chemicals and pathogens respectively.

Drinking Water Microbiology

Usage of

Water Bacteriology

Determining whether a water supply is fecally polluted is to test for the presence of normal fecal organism.

Testing for normal fecal organisms as indicators of fecal pollution is a reliable way of determining whether water is bacteriologically safe to drink.

A single laboratory examination of any water does not justify the conclusion that the supply is safe for drinking so bacteriologic analysis of water should be performed at regular intervals.

This analysis is used in companies that manufactured water supplies like water bottles, the ones we get on fast food restaurants and stores. Namely: Nature Spring, Wilkins, Absolute, Summit etc.

With this analysis, one can be sure that the water we drink is safe and free from microorganisms. So, one can also prevent getting diseases from microbes that can live on water especially the fecal coliforms.

Water-borne diseases

• An important aspect of Water Microbiology is numerous disease causing microorganisms spread through water.

• Many bacteria, viruses, fungi and protozoa are responsible for waterborne diseases.

• The recurrence of waterborne illness has led to the improvement in water purification.

Potable - (clean) water – free of all objectionable material, including pathogens, tastes, odors, colors, toxins, radioactive material, organisms, oils, gases, etc.

Fresh – non-salt or sea water Pollution – anything that makes it Non-

Potable Sewage – the community waste or garbage

that mother nature and we dump onto sewers or land

Terminology

Typical Water Quality Standards

Drinking Water◦ No coliforms contamination

acceptable Recreational water

◦ 200 fecal coliforms /100 ml Fish and wildlife habitat

◦ 5000 fecal coliforms/100 ml Shellfish

◦ 14 fecal coliforms/100 ml

Indicator Organisms General coliforms –

indicate water in contact with plant or animal life (universally present)

Fecal coliforms – mammal or bird feces in water

Enterococcus bacteria (type of fecal streptococci)– feces from warm blooded animals in water

These are not what generally make people sick

Bacteria whose natural habitat is water are not well known, partly due to the fact that many of them are difficult to grow on laboratory media. The group includes the following:

1.higher bacteria- sulfur bacteria, iron bacteria

2.appendaged bacteria attached to some ininanimate objects

3.large spiral forms

Bacteria native to natural waters

4. a variety of bacilli -Pigmented form- Serratia marcescens,

Chromobacterium violaceum -Non pigmented form-Pseudomonas flourescens.

Thermophils, arerobid and anaerobic sopreformers

5.Coccus forms6. Nitrogen fixing bacteria7.Nitrifying bacteria

A. bacteria from air and soil the number of bacteria in the air bears a

close relation to the amount of larger suspended particles of dust and which fluid their way into natural water through settling out or are swept down by the rain

soil contamination contain tremendous numbers of bacterria most of which are found un the upper 6 inches of the soil

Bacterial contamination of natural waters

The others are present as contaminaton coming from:

1. Excreta of humans and aninmals2. Flesh of animas and humans who have

died of infectious diseasesB.Direct contamination of water by human

and animal excreta-noemal intestinal flora which maybe found in

water3. Large number of coliform bacilli4. Clostridium perfringens5. Alpla-hemolytic fecal streptococci or

enterococci

-pathogenic organisms shed from feces of

infected persons and carriers iinclude:1.bacterial enteric pathogens2. viral souce

Bacteria Disease/ infection Symptoms Aeromonas Enteritis Very thin, blood- and

mucus-containing diarrhea

Campylobacter jejuni Campilobacteriose Flue, diarrhea, head- and stomachaches, fever, cramps and nausea

Escherichia coli Urinary tract infections, neonatal meningitis, intestinal disease

Watery diarrhea, headaches, fever, homiletic uremia, kidney damage

Plesiomonas shigelloides Plesiomonas-infection Nausea, stomachaches and watery diarrhea, sometimes fevers, headaches and vomiting

Typhus Typhoid fever Fevers

  Salmonella

Salmonellosis Sickness, intestinal cramps, vomiting, diarrhea and sometimes light fevers

Streptococcus (Gastro) intestinal disease Stomach aches, diarrhea and fevers, sometimes vomiting

Vibrio El Tor (freshwater) (Light form of) Cholera Heavy diarrhea

1.Bacteria Found In Surface Water

2.Viral Sources of Waterborne Disease Hepatitis A: inflammation

and necrosis of liver Norwalk-type virus: acute

gastroenteritis Rotaviruses: acute

gastroenteritis, especially in children

Enteroviruses: many types affect intestines and upper respiratory tract

Reoviruses: infects intestines and upper respiratory tract

Other indicator organisms

Indicator organism

Characteristics Significance

Clostridium perfringens

anaerobic spore former, gram positive rod shaped and exclusively of fecal origin. Spores are resistant and persist for long periods.

useful indicator of past pollution, a tracer for less hardy indicators, protozoans and viruses.

Bifidobacterium and Bacteroids

primarily associated with humans they can distinguish human and animal contamination.

B. bifidussurvives for a short time therefore its presence suggests relatively recent pollution

Indicator organism

Characteristics Significance

Pseudomonas aeruginosa

associated with the diseases of eye, ear, nose and throat infections. common opport-unistic pathogen, causes life threatening infection in burn patients and immuno-compromised individuals. Folliculitis, dermatitis, ear and urinary infections are common in ill maintained swimming pools.

this organism is of no value as indicator of fecal pollution however coliforms do not suit as indictor of contamination of swimming pool water as the contamination is not of fecal origin.

Indicator organism

Characteristics Significance

Staphylococcus aureus and Candida albicans

suggests the sanitary quality of water because it presence is associated with human activities

Useful for recreational waters.

Aeromonas hydrophila

occurs in uncont-aminated, as well as contaminated waters. also an opportunistic pathogen in humans, animals and fish.

Because of its association with nutrient rich conditions it has been suggested as an indicator of nutrient rich status of the waters.

A. Type of water1.surface water: streams, lake, shallow well

-oppurtunities for contamination are many bacterias are present

2.water from deep driven wells:water has undergone effective filtration in order to reach the surface strata. Hence this only contain a few number of bacteria

B. Amount of organic matter present:-more nutrient there is pressent in the form of organic matter, the greater amount of bacteria

C. Temperature-low temperature are not conducive to rapid growth; higher temperatures result in a increased bacterial nimbers in the present of sufficient organanic matter

Factors affecting the kinds and numbers of bacteria

Numerous water borne pathogens Individual pathogen numbers may be too

low to detect in a reasonable sized water sample

Isolation and detection of some pathogens can take several days, weeks, or months

Absence of one particular pathogen does not rule out the presence of another

Microbiological Examination of Water

Methods of bacteriologic analysis of water sample

Correlated to the presence of pathogens Population large enough to isolate in

small water samples (100 mL) Rapid Inexpensive Safety, not culturing pathogens

Indicator Organism Concept

Enterobacteriaceae◦Facultative anaerobe◦Gram negative◦Non-spore forming◦Rod shaped◦Ferment lactose◦Produce gas and acid

within 48 h @ 35ºC

Coliform genera◦Enterobacter◦Klebsiella◦Citrobacter◦Escherichia

Coliform Group (total coliform)

I. Presumptive Test -Label: 3 tubes of Lauryl Tryptose Broth (10ml)

3 tubes (1.0ml) 3 tubes (0.1ml)

-Shake the bottle with water sample vigorously (25x) and transfer 10ml of water to the first 3 tubes (10ml)

transfer 1.0ml of water sample to the next tubes (1.0ml)

transfer 0.1ml water sample to the last 3 tubes (0.1ml)

A. Multiple Fermentation Technique

- Incubate @ 37°C for 24 hrs -After 24 hrs, observe for the presence of gas in the

inverted vials. -No gas = re-incubate for another 24 hrs and re-

examine -Record the presene or absence of gas

-Record the number of tubes giving the (+) gas production result

II. Confirmed Test -Carefully shake each (+) tube in the Presumptive Test -Transfer 1 loopful of the culture to BGLB and EC

tubes. -Agitate the tubes to mix the inoculum -Incubate the BGLB tubes @ 37°C and EC tubes @

44°C in the water bath for 24 hrs

-After 24 hrs incubation, examine for gas production -Any amount of gas present in BGLB and EC tubes =

(+) Confirmatory Test -No gas= re-incubate for another 24 hrs -After 48 hrs incubation, re-examine BGLB tubes -Record the presence or absence of gas -Discard tubes without gas and keep the tubes with

gas for the Completed Test

III. Completed Test -Streak an EMB plate from each tube of BGLB with

gas -Incubate plates @ 37°C for 24 hrs -Pick one or more typical well isolated coliform

(colored) colonies. If no typical colonies are present, pick 2 or more colonies considered most likely to consist of organisms of the coliform group

-Transfer growth from each isolated colony to Lauryl Tryptose Broth fermentation tube and to Nutrient Agar slant

-Incubate Lauryl Tryptose Broth @ 37°C for 24 hrs and observe for gas production

-No gas = re-incubate for another 24 hrs and re-examine for gas production

-Make Gram Stained smears of colonies from NA slant and look for Gram (-) nonsporing coccobacilli

Label 1 petri dish 1ml & another petri dish 0.1ml Mix water sample thoroughly by shaking vigorously

(25x) Place 1ml water sample = 1ml petri dish and 0.1ml

water sample =0.1ml petri dish Add to each plate 10ml liquefied NA cooled 42-45°C Mix the agar and water by tilting and rotating the

plates, allow to solidify Incubate @ 37°C for 24 hrs Colony Counting: Select the plate in which colonies are evenly

distributed and containing 30-300 colonies

B. Total Bacterial Count:

Count all the colonies including those pinpoint size

Use Quebec Colony Counter Calculate the colonies per cubic centimeter by

multiplying the number of colonies in the plate by the fraction of cubic centimeter of sample used

Serial dilution to extinction Inoculate multiple tubes (5 or 10) of media

with across the increasing series of dilutions Incubate

◦ 35ºC or ◦ 44.5ºC

Count positive growth tubes Use Most-Probable-Number (MPN) table to

estimate density

Multiple Tube Fermentation Methods

INTERPRETATION OF RESULTS

Formation of at least 10% gas in the inner fermentation tubes or vials within 48 hours (positive test)

If gas is formed, the broth medium will become cloudy

Active fermentation may be shown by the continued presence of small bubbles of gas throughout the medium outside inner vial

PRESUMPTIVE TEST

Absence of gas formation at the end of 48 hours of incubation constitutes a negative result

Submit all primary fermentation tubes showing any amounts of gas within 24 hours of incubation to the Confirmed Test

If active fermentation appears in the primary fermentation tubes earlier than 1 hour,

preferably transfer to the confirmatory medium without waiting for full 24 hours

If additional primary fermentation tubes show gas production at the end of 24 hours, submit them to the Confirmed Test.

Formation of gas in inverted vial of BGLB fermentation tube at any time within 48 hours constitutes a positive Confirmed Test.

Gas production in the EC fermentation tube within 24 hours or less is a positive reaction indicating fecal origin.

Failure to produce gas is a negative reaction

CONFIRMED TEST

The Most Probable Number (MPN) of coliform organisms present can be determined based on the number of positive tubes obtained in the Confirmatory Test (BGLB tubes)

Based on the British Ministry of Health, waters are divided into classes on the following basis:

CLASS I – regarded as highly satisfactory and contains less than 1 coliform/100mL

CLASS II – regarded as satisfactory and contains 1-2 coliform/100mL

CLASS III – regarded as suspicious and contains 3-10 coliforms/100mL

CLASS IV – regarded as unsatisfactory and contains more than 10 coliforms/100mL

The colonies developing on EMB are called typical or atypical after 24 hours of incubarion

Formation of gas in the second tube of LTB within 48 hours and demonstration of gram (-) nonsporeforming rod-shaped bacteria in the NA slant constitute a satisfactory Completed Test demonstrating the presence of a member of the coliform group.

COMPLETED TEST

The analysis consists in counting the organisms present in a given unit volume of water on a standard agar medium incubated at 37 degrees Celsius for 24 hours

It is not a true total count for it misses dead bacteria and those that do not grow at 37 degrees Celsius and bacteria that does not form visible colonies within 24 hrs. under standard condition.

BACTERIAL COUNT

It should be reported either as the number of colonies developing per mL or simply plate count per mL.

Filter water through a 0.45 μM membrane filter

Place membrane on selective media Incubate

◦ 35ºC total coliform◦ 44.5ºC fecal coliform

Count colonies

Membrane Filter Methods

Water purification forms a critical link in promoting public health and safety.

It involves variety of steps which depend upon the type of impurities in the raw water source.

The major operations done are sedimentation, flocculation, filteration and disinfection.

Water purification

1.Mechanical methods:1.1 storage1.2 filtration

1.2.1 slow sand filtration1.2.2 coagulation and rapid sand filtration

2. chemical methods:2.1 large scale: hypocholrite and liquid chlorine2.2 small scale:hypochlorite, UV light , Iodine

Unit Processes / operations Effect 1 Aeration, chemical oxidation, ion

exchange, sedimentation Colour and precipitate removal

2 Chemical precipitation, (dosing, mixing, flocculation, settling) ion exchange

Softening (Ca, Mg removal)

3 Chemical coagulation, (dosing, mixing, flocculation, settling) filtration

Turbidity removal

4 Aeration, chemical oxidation, adsorption Taste and odour removal

5 Irradiation, ozonation, chlorination Disinfection

Unit processes and operations and specific impurities removed

Bottom Line Test your water as required and

anytime you suspect a problem

Work with your County Environmental Health Department

Seek advise on interpreting the results – what do they mean?

If you question the results, resample and retest

References:http://www2.fiu.edu/~psharp01/Exp9WaterBact.pdfhttp://www.slideshare.net/HamaNabaz/water-microbiology-21923074