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

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.

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