Exp 4 Total Fecal Coliform

Experiment #  To determine total coliform and fecal coliform by multiple tube fermentation technique.

Total coliforms

The Coliform group comprises all aerobic and facultative anaerobic, gram negative, non spore forming, rod shaped bacteria that ferment lactose with gas and acid formation within 48 hours at 350C.

Purpose

  • The most common risk to human health associated with water stems from the presence of disease causing micro-organisms (Pathogens). Many of these pathogens originate from water polluted with human excrements. Intestinal bacteria pathogens are distributed world wide;
  • The purpose of examination of no potable water generally is to estimate the density of bacteria contamination, determine a source of contamination, or trace the survival of micro-organism. Each objective requires a numerical value for reporting results. The multiple tube fermentation technique may be used to obtain statistical valid MPN estimate of the Coliform;
  • The objective of Coliform test is to determine compliance with EPA standards as a measure of the efficiency of treatment plant operation or water effluent quality. A high proportion of Coliform occurrence in the distribution system may be attributed not to treatment failure at the plant or the well source, but the bacterial re-growth in the mains.

Limitations

Carefully observe specified time for Coliform because further proceeding is required in the presence of positive results. It is very sensitive test so neighboring contamination may also effect on the results.

Sampling and Storage

  • Strong thick walled glass or plastic bottles free of contamination should be used for collecting samples for microbiological analysis.
  • Samples whose transportation require more than 6 hours but less than 24 hours, such bottles should be rapidly chilled rat about 40C immediately after collection and kept during transportation.
  • Sample arriving more than 24 hours after sampling should be discarded.

Standard Presumptive Method

Lactose broth may be used as an alternative medium provided that it has been demonstrated not to increase the frequency of false positive nor mask Coliform present in drinking water samples. If the medium has been refrigerated after sterilization, incubate overnight at 350C before use. Discard tubes showing growth and/or bubbles.

Apparatus

  • Autoclave
  • Incubator
  • Sample bottles
  • Fermentation tubes with inverted vials
  • Dilution bottles
  • Pipettes and pipette stand

Reagents

Lactose broth

Distilled water

Procedure

  • For potable water arrange ten fermentation tubes in rack with inverted vials. Before sterilization, dispense sufficient medium, to cover inverted vials at least partially after sterilization;
  • Sterilize the fermentation tubes containing the medium along with other necessary glass apparatus in an autoclave for 15 minutes at 1210C;
  • Remove fermentation tubes from autoclave as soon as the chamber pressure reaches to zero. Never re-autoclave the medium;
  • Dispense 10 mL of sample in each tube and incubate inoculated tubes at 35 ± 0.50C. After 24 ± 2 hours shake each tube gently and examine it for gas or acidic growth. If no gas or acidic growth has formed, re-incubate and re-examine at the end of 48 ± 2hrs. Record the presence or absence of gas or acid production in the fermentation tubes;
  • Absence of acidic growth or gas formation at the end of 48 ± 3 hours of incubation constitute a negative test;
  • Production of gas or acidic growth in the tubes within 48+3 hours constitutes a positive presumptive reaction. Submit these tubes with a positive presumptive reaction to the confirmed phase;
  • Shake sample and dilutions vigorously about 25 times and repeat same procedure mention as for portable or drinking water.

Confirmed Phase

Reagent

  • Brilliant Green Lactose Bile Broth (BGLB)
  • Distilled Water

Apparatus

  • Fermentation tubes with caps
  • Inverted vials
  • Sterile metal loop 3 mm in diameter
  • Sprit Lamp

Procedure

  • Before sterilization, dispense sufficient medium, to cover inverted vials at least partially after sterilization;
  • Submit all primary tubes showing any amount of gas or acidic growth with in 24+2 or 48+3 hours of incubation to the confirmed phase;
  • If active fermentation or acidic growth appears in the primary tubes earlier than 24 hours, transfer to the confirmatory medium, preferably without waiting for the full 24+2 hours period to elapse;
  • If additional primary tubes show acidic growth at the end of a 48+3 hours incubation period, submit these to the confirmed phase;
  • Gently shake or rotate primary tubes showing gas or acidic growth to re-suspend the organisms.
  • Take a metal inoculating loop of 3 mm diameter and heat it on the sprit lamp till it becomes red-hot;
  • Cool the loop to room temperature and with its help transfer one loop full of culture to a fermentation tube containing brilliant green lactose bile broth.
  • Incubate the inoculated Brilliant Green Lactose Bile Broth tube for 48 ± 3 hours at 35 ± 0.50C.
  • Formation of gas in any amount in the inverted vial of the brilliant green lactose bile broth fermentation tube at any time with in 48 ± 3 hours constitute a positive confirmed phase.
  • Calculate the MPN value from the number of positive brilliant green lactose bile tubes as

MPN/100ml = No of Positive Tube x 100/√(ml of sample in negative tube)(ml of sample in all tubes

exp 7

FECAL COLIFORM

Definition

This group comprises the Coliform Bacteria whose origin is feces (intestines of warm-blooded animals). This test differentiates between Coliforms of fecal origin and Coliforms of non-fecal origin.

Purpose

  • The most common risk to human health associated with water stems from the presence of disease causing micro-organisms (Pathogens). Many of these pathogens originate from water polluted with human excrements. Intestinal bacteria pathogens are distributed world wide;
  • The purpose of examination of no potable water generally is to estimate the density of bacteria contamination, determine a source of contamination, or trace the survival of micro-organism. Each objective requires a numerical value for reporting results. The multiple tube fermentation technique may be used to obtain statistical valid MPN estimate of the Coliform;
  • The objective of Coliform test is to determine compliance with EPA standards as a measure of the efficiency of treatment plant operation or water effluent quality. A high proportion of Coliform occurrence in the distribution system may be attributed not to treatment failure at the plant or the well source, but the bacterial re-growth in the mains.

 

Limitations

Carefully observe specified time for Coliform because further proceeding is required in the presence of positive results. It is very sensitive test so neighboring contamination may also effect on the results; During inoculation from presumptive phase take an extra care to ensure that bacteria have been transferred to new medium.

Sampling and Storage

  • Strong thick walled glass or plastic bottles free of contamination should be used for collecting samples for microbiological analysis.
  • Samples whose transportation require more than 6 hours but less than 24 hours, such bottles should be rapidly chilled rat about 40C immediately after collection and kept during transportation.
  • Sample arriving more than 24 hours after sampling should be discarded.

Reagents

  • EC medium
  • Distilled Water

Apparatus

  • Fermentation tubes with caps
  • Inverted vials
  • Sterile metal loop 3 mm diameter
  • Sprit Lamp

Procedure

  • Before sterilization dispense in fermentation tubes each with an inverted vial sufficient medium to cover the inverted vial at least partially after sterilization. Close tubes with caps;
  • Sterilize the tubes containing medium and other necessary glassware at 1210C for 15 minutes in an autoclave;
  • Submit all presumptive fermentation tubes showing any amount of gas or heavy growth with in 48 hours of incubation to the confirmed test;
  • Gently shake or rotate presumptive fermentation tubes showing gas or heavy growth;
  • Take a 3 mm diameter metal loop and heat to red-hot on the sprit lamp;
  • Cool the loop to room temperature and with the help of this loop transfer growth from each presumptive fermentation tube to EC broth;
  • Incubate inoculated EC broth tubes at 44.5 ± 0.20C for 24 ± 2 hours;
  • Gas production in an EC broth culture with in 24 hours or less is considered a positive fecal Coliform reaction;

Failure to produce gas (growth sometimes occurs) constitute a negative reaction indicating a source other than the intestinal trace of a warm-blooded animals.

exp 7

OBSERVATIONS AND CALCULATIONS

Lactose Brothe (PresumtiveTest)

Sr No

Volume of Sample Used

Total Tubes

Lactose Brothe Solution

Positive Tubes

Negative Tubes

1

10 ml

5

10ml

4

1

2

1ml

5

10ml

2

3

3

0.1 ml

5

10ml

3

2

BGBB (Total Coliform)

Sr No

Volume of Sample Used

Total Tubes

BGBB Solution

Positive Tubes

Negative Tubes

1

10 ml

5

10ml

3

2

2

1ml

5

10ml

2

3

3

0.1 ml

5

10ml

3

2

Total Volume of Sample  = 55.5 ml

Volume of Positive test tubes = 32.3 ml

Volume of Negative test tubes = 23.2 ml

Total Coliform  = 22 MPN / 100 ml

E.C. Brothe (Fecal Coliform)

Sr No

Volume of Sample Used

Total Tubes

BGBB Solution

Positive Tubes

Negative Tubes

1

10 ml

5

10ml

2

3

2

1ml

5

10ml

1

4

3

0.1 ml

5

10ml

0

5

Total Volume of Sample  = 55.5 ml

Volume of Positive test tubes = 21.0 ml

Volume of Negative test tubes = 34.5 ml

Fecal Coliform  = 7 MPN / 100 ml

Question Answers

1)    What  are bacteria?

Bacteria: Single-celled microorganisms which can exist either as independent (free-living) organisms or as parasites (dependent upon another organism for life).

Examples of bacteria include:

  • Acidophilus, a normal inhabitant of yogurt,
  • Chlamydia, which causes an infection very similar to gonorrhea,
  • Clostridium welchii the most common cause of the dreaded gas gangrene,
  • E. coli, the common peaceful citizen of our colon and, upon occasion, a dangerous agent of disease, and
  • Streptococcus, the bacterium that causes the important infection of the throat strep throat.

The term bacteria was devised in the 19th century by the German botanist Ferdinand Cohn (1828-98) who based it on the Greek bakterion meaning a small rod or staff. In 1853, Cohn categorised bacteria as one of three types of microorganisms — bacteria (short rods), bacilli (longer rods), and spirilla (spiral forms). The term bacteria was preceded in the 17th century by the microscopic animalcules described by Antony van Leeuwenhoek (1632-1723).

2)    What is the health based guideline value for Total coliform and fecal coliform group?

Coliform bacteria are generally not harmful to human health however the total coliform group includes fecal coliforms and E. coli. As such, the presence of coliform organisms can indicate the possibility of pollution by human or animal waste. Health based guideline for Total coliforms is 0 organisms/100mL . Must not be detectable in any 100ml sample
(Each person discharges from 100 to 400 billion fecal coliform organisms per day). Table 1. Maximum Allowable limits of fecal coliform bacteria

3)    Compare bacteria with aquatic plants?

Aquatic plants promote bacterial growth by releasing added oxygen into the soil, by excreting organic compounds into sediments and by providing microorganisms with a support on which they can multiply.

4)    Name some diseases caused by bacteria.

Disease

Casual Agent

Description of Agent

Organs Affected

Transmission

Strep Throat,Scarlet Fever

Streptococcus, Pyogenes

Gm(+) capsualted streptococcus

Upper respiratory tract

Air

Diphtheria

Corynebacterium diphtheriae

Gm(+) rod

blood,Skin, Upper Respiratory tract,Heart,Nerve Fibres

Air

Pertussis ( Whooping Cough)

bordetella pertussis

Gm (-) rod

Upper Respiratory Tract

Air

Meningococcal Meningitis

Neisseria meningitidis

Gm(-) diplococcus

Upper Respiratory Tract, Blood, Meninges

Air

Haemophilus meningitis

Haemophilus influenzae

Gm(-) capsulated rod

Upper Respiratory Tract, Meninges

Air

Flavobacterium meningitis

Flavobacterium meningospecticum

Gm(-) rod

Upper Respiratory Tract,Meninges

Air

Tuberculosis

Mycobacterium tuberculosis

Acid fast rod

Lungs, Bonesm Other Bones

Air

Pneumococcal pneumonia

Sterptococcus pneumoniae

Gm(+) capsualted diplococci in chains

Lungs

Air

Primary Atypical Pneumonia

Mycoplasma pneumoniae

Mycoplasma ( No Cell Wall )

Lungs

Air

Klebsiella pneumonia

Klebsiella pneumoniae

Gm(- ) capsulated rod

Lungs

Air

Serratia pneumonia

Serratia marcescens

Gm(- ) rod, Red pigement at 25 degree C

Lungs

Air

Q Fever

coxiella burnetti

Rickettsia, 0.45 micron Diameter

Lungs

Air

Psittacosis

chlamydia psittaci

Chlamydia, 0.25 micron diameter

Lungs

Air

Botulism

Clostridium botulinum

Gm(+) spore forming rods

Neuromuscular Junction

Food, Water

Staphylococcal Food Poisoning

Stephylococcus aureus

Gm(+) Staphylococcus

Intestine

Food, Water

Clostridial Food Poisoning

clostridium perfringes

Gm(+) Spore forming Rods

Intestine

Food, Water

Typhoid Fever

Salmonella typhi

Gm(-) rod

intestine, Blood, Gall Bladder

Food, Water

Salmonellosis

Salmonella serotypes

Gm(-) rods

Intestine

Food, Water

Shigellosis

shigella serotypes

Gm(-) rods

Intestine

Food, Water

Cholera

vibrio cholerae

Gm(-) Curves rod

Intestine

Food, Water

Brucellosis

Brucella Spp.

Gm(-) rod

Spleen,Lymph Glands

Food, Water

Anthrax

Bacillus anthracis

Gm(+) Spore Forming Rod

Blood, Lungs,Skin

Soil

Tetanus

Clostridium tetani

Gm(+) spore forming anaerobic rod

Nerves at synapse

Soil

Gas Gangrene

clostridium perfringes

Gm(+) spore forming anaerobic rod

Muscles,Nerves, Blood Cells

Soil

Bubonic Plague

yersinia pestis

Gm(-) bipolar rod

Lymph Nodes,Blood,Lungs

Rat flea (A)

Relapsing Fever

Borrelia recurrentis

Spirochete

Blood, Liver

Louse (A)

Rocky Mountain Spotted Fever

Rickettsia rickettsiae

Rickettsia

Blood, Skin

Tick (A)

Epidemic Typhus ( Typhus Fever)

Rickettsia prowazekii

Rickettsia

Blood, Skin

Louse (A)

Endemic Typhus ( Murine Typhus)

Rickettsia typhi

Rickettsia

Blood, Skin

Flea (A)

Scrub Typhus

Rickettsia tsutsugamushi

Rickettsia

Blood, Skin

Mite (A)

Rickettsialpox

Rickettsia akari

Rickettsia

Blood, Skin

Mite (A)

Tickborne Fevers

Rickettsia conorii

Rickettsia

Blood, Skin

Tick (A)

syphilis

Treponema pallidum

Spirochete

Skin, Cardiovascular Organs

Sexual

Gonorrhea

Neisseria gonorrhoeae

Gm(-) diplococcus

Urethra,Cervix,Fallopian Tubes, Epididymis, Eyes, Pharynx

Sexual

Chlamydial urethritis

chlamydia trachomatis

chlamydia

Urethra,Cervix,Fallopian Tubes, Epididymis, Eyes, Pharynx

Sexual

Ureaplasmal urethritis

Ureaplasma urealyticum

Mycoplasma

Urethra,Fallopian tubes,Epididymis

Sexual

Lymphogranuloma venereum

Chlamydia trachomatis

Chlamydia

Inguinal lymph nodes,Rectum

Sexual

Vaginitis

Gardnerella vaginalis

Gm(-) rod

Vagina

Sexual

Mycoplasmal urethritis

Mycoplasma hominis

Mycoplasma

Urethra, Fallopian tubes

Sexual

Leprosy ( hansen’s Disease)

Mycobacterium leprae

Acid Fast Rod

Epididymis skin,bones,periphercal nerves

Contact

Staphylococcal skin diseases

Staphlococcus aureus

Gm(+) staphylococcus

skin

Contact

Trachoma

chlamydia trachomatis

Chlamydia

Eyes

Contact

Bacterial Conjuctivitis

Haemophilus influenze type III

Gm(-) rod

Eyes

Contact

5) What  are indicator  Organisms?

These are microbes whose presence in water signals the presence of fecal matter, and potentially, pathogens. One such indicator organism is Escherichia coli, a bacterium that is normally found in the guts of humans and other warm-blooded animals. Water that is highly polluted with fecal matter may have E. coli counts in the tens of millions of bacteria per liter. In the State of Michigan, beaches are ordered closed if the E. coli count exceeds a monthly average of 130 or a maximum of 300 bacteria per 100 milliliters. A drinking water source is declared safe only if these bacteria are not detected in the sample.

Indicator organisms are a basic monitoring tool used to measure both changes in environmental water quality or conditions, and the potential presence of hard-to-detect pathogenic organisms.  An indicator organism provides evidence of the presence or absence of a pathogenic organism that survives under similar physical, chemical, and nutrient conditions.

It is important to note – an indicator is not necessarily a pathogen.  Although some strains of E. coli are pathogenic, the reasons E. coli and Enterococci are used are, because they have been shown to be indicative of recent fecal contamination.  In addition, their behavior (viability, longevity, movement) in the environment is assumed to be similar to actual pathogens of concern, and there is a relatively fast method of analysis available. Suggested Indicator Organisms

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