IDENTIFICATION, ISOLATION AND CULTURING OF HUMAN INTESTINAL E.

COLI

ANUPAMA CN 08CBT4057

IDENTIFICATION, ISOLATION AND CULTURE OF HUMAN INTESTINAL E.COLI

Dissertation submitted to St. Josephs College in partial fulfillment of the bachelor of science course in chemistry, botany and biotech

Submitted by Anupama CN 08CBT4057 Guided by Dr. GRACE PRABHAKARAN Department of Biotechnology

St. Josephs College of Arts and Science (autonomous), Bangalore

ST.JOSEPHS COLLEGE BANGALORE

CERTIFICATE
This is to certify that this project entitled IDENTIFICATION, ISOLATION AND CULTURE OF HUMAN INTESTINAL E.COLI is a bonafide work done by Anupama CN. This project work was done during the year 2010-11 in the biotechnology laboratory of the Under Graduate department, under the supervision of Dr. GRACE PRABHAKARAN, Department of Biotechnology, St. Josephs College of Arts and Science (Autonomous), Bangalore.

Dr.Grace Prabhakaran Dept. of Biotechnology Date:

Principal

DECLARATION
I hereby declare that this study on IDENTIFICATION, ISOLATION AND CULTURE OF HUMAN INTESTINAL E.COLI is a bonafide work and has been prepared under the supervision of Dr. GRACE PRABHAKARAN, Department of Biotechnology, Josephs College (Autonomous), Bangalore, at Biotechnology laboratory of the Under Graduate department of St. Josephs College of Arts and Science.

Anupama CN

ACKNOWLEDGEMENT
It gives me an immense pleasure to acknowledge all the people who helped me to complete this project. My sincere thanks to Fr.Ambrose Pinto, Principal, St.Josephs college for providing me an opportunity and all the necessary facilities to carry out my project work. I am indebted to Dr. Grace Prabhakaran, my project guide for her guidance, constant encouragement and all the facilities kindly provided to me during the project. I also thank and Mrinmoy Sarkar, my work partner for his cooperation and unending encouragment in successful completion of my project work. And last but not the least, I would like to thank CN Ramesh, my father, for all the support he lent to me through out the completion of my work, not just as a parent but also a mentor. I would also like to thank him specially for his constructive criticism.

Anupama CN

CONTENTS
CHAPTER
INTRODUCTION PROCEDURE Media Preparation Sampling of Intestinal Microflora Culturing of Intestinal Microflora Isolation of E.coli Culturing of E.coli CONCLUSION

PAGE No.
6-10 11-14 15-16 17-18 19-20 21-25 26

INTRODUCTION
INTESTINAL MICROFLORA
Intestinal flora are harmless microorganisms such as bacteria, yeasts, and fungi that inhabit and grow in the intestines. These microorganisms are essential to the normal functioning of the digestive tract, and certain species of intestinal flora are beneficial to the human body. These good bacteria often have a symbiotic relationship with the human body as both derive benefit from one another. For example, The normal [intestinal] flora derive from their host a steady supply of nutrients, a stable environment, and protection and transport. The host obtains from the normal [intestinal] flora certain nutritional and digestive benefits, stimulation of the development and activity of immune system, and protection against colonization and infection by pathogenic microbes. Beneficial bacteria such as intestinal flora are often referred to a probiotics. Probiotics is an umbrella term given to any live microorganism that is beneficial to its host.

LIST OF INTESTINAL MICROFLORA OR PROBIOTICS

Lactobacillus acidophilus These are the most friendly bacteria.They are naturally present in dairy products and also added in dietary supplements for better results. Tobacillus acidophilus: They are acid loving bacteria. These beneficial bacteria, found in buttermilk, yogurt, sour cream and frozen desserts, convert sugar and carbohydrates into lactic acid and so, are called lactic acid bacteria. By lowering the pH and reducing the risk of growth of other organisms in the food through this

process, they generate the fermented taste. This process is beneficial for people, as it prevents the gastrointestinal infections.

Cyanocobalamin These bacteria help in the production of vitamin B12 during the process of digestion. Probiotics are responsible for the overall digestive health, as they break down the complex components of the food, so that, they can be easily absorbed in the blood.

Acidophilus bifidus Acidophilus bifidus reduce cholesterol levels and prevent the growth of hostile yeasts like Candida albicans. The acidophilus bifidus bacteria cleanse the blood stream by removing the toxins. Thus, they enhance the immune system.

Streptomyces Streptomyces are used in manufacturing of antibiotics.

Rhizobium These play an important role in nitrogen fixation.

E. Coli - Escherichia Coli Bacteria These are present in the intestine and are also known as enteric bacteria. These bacteria help in digestion and keep us healthy. They also produce vitamin B-complex and vitamin K.

Saccharomyces boulardii Saccharomyces boulardii help to reduce the risk of antibiotic-associated diarrhea in children.

Anaerobic Beneficial Bacteria

Anaerobic beneficial bacteria are helpful in fermentation of vinegar and in the cheese making process too.

E.COLI
E. coli was discovered by German paediatrician and bacteriologist Theodor Escherich in 1885,[and is now classified as part of the Enterobacteriaceae family of gamma-proteobacteria. Escherichia coli (commonly abbreviated E. coli) is a Gram-negative rodshaped bacterium that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some can cause serious food poisoning in humans. The harmless strains are part of the normal flora of the gut (intestine) and can benefit their hosts by producing vitamin K 2, and by preventing the establishment of pathogenic bacteria within the intestine. E. coli are not always confined to the intestine, and their ability to survive for brief periods outside the body makes them an ideal indicator organism to test environmental samples for fecal contamination. The bacteria can also be grown easily and its genetics are comparatively simple and easily manipulated or duplicated through a process of metagenics, making it one of the best-studied prokaryotic model organisms, and an important species in biotechnology and microbiology.

E.COLI

Role in disease
Virulent strains of E. coli can cause gastroenteritis, urinary tract infections, and neonatal meningitis. In rarer cases, virulent strains are also responsible for haemolytic-uremic syndrome, peritonitis, mastitis, septicaemia and Gramnegative pneumonia. Certain strains of E. coli, such as O157:H7, O121 and O104:H21, produce potentially lethal toxins. Food poisoning caused by E. coli is usually caused by eating unwashed vegetables or undercooked meat. O157:H7 is also notorious for causing serious and even life-threatening complications such as Hemolytic-uremic syndrome. This particular strain is linked to the 2006 United States E. coli outbreak due to fresh spinach. Severity of the illness varies considerably; it can be fatal, particularly to young children, the elderly or the immune compromised, but is more often mild. Earlier, poor hygienic methods of preparing meat in Scotland killed seven people in 1996 due to E. coli poisoning, and left hundreds more infected. E. coli can harbour both heat-stable and heatlabile enterotoxins. The latter, termed LT, contains one A subunit and five B subunits arranged into one holotoxin, and is highly similar in structure and function to cholera toxins. The B subunits assist in adherence and entry of the toxin into host intestinal cells, while the A subunit is cleaved and prevents cells from absorbing water, causing diarrhea If E. coli bacteria escape the intestinal tract through a perforation (for example from an ulcer, a ruptured appendix, or due to a surgical error) and enter the abdomen, they usually cause peritonitis that can be fatal without prompt treatment. However, E. coli are extremely sensitive to such antibiotics as streptomycin or gentamicin. This could change since, E. coli quickly acquires drug resistance. Recent research suggests that treatment with antibiotics does not improve the outcome of the disease , and may in fact significantly increase the chance of developing haemolytic-uremic syndrome. Intestinal mucosa-associated E. coli are observed in increased numbers in the inflammatory bowel diseases, Crohn's disease and ulcerative colitis. Invasive strains of E. coli exist in high numbers in the inflamed tissue, and the number of bacteria in the inflamed regions correlates to the severity of the bowel inflammation.

STEP 1

MEDIA PREPARATION
SELECTION OF MEDIA
E.coli being a bacteria can grow easily in many growth media. Minimal liquid media, rich liquid media, solid media, top agar and stab agar are the different kinds of media on which E.coli can grow. Essentially it needs tryptone, yeast extract, agar (Bacto-agar) and nutrient broth to grow. But when using any media, the following tests have to be conducted on all the colonies of the sample showing positive for E.coli to differentiate E.coli.

Table 1: Culture characteristics of E.coli on different media

Media used
Mac Conkey Agar Smooth

Culture character
circular pink colonies with spreading growth. Non hemolytic, grey white moist, glistening, opaque, circular, convex colonies with entire edge. Colorless and yellowish white, circular, smooth colonies with entire edge. Organism showed uniform turbidity. Small, circular pink colonies.

Blood Agar

Nutrient Agar

Nutrient Broth Violet Red Bile Agar

Table 2: Biochemical reactions of E.coli Biochemical Test

Catalase Simmons citrate TSI Gelatin liquefaction Indole Production Nitrate Reduction Urease Voges Proskaur Methyl Red Presumptive test

Reaction
+ve -ve A/A+gas -ve +ve +ve -ve -ve +ve +ve

This process is time consuming and also becomes very tedious. Hence we use a media that is highly specific and developed for the differentiation of Escherichia coli. Its is the Eosin Methylene Blue Agar (which from here on will be reffered to as EMB agar). Using this media, it becomes very easy to identify E.coli colonies by mere observation itself.

 PREPARATION OF MEDIA
EMB Agar (Eosin Methylene Blue Agar)
EMB Agar is a very versatile solid medium. Originally developed by Levine for the differentiation of Escherichia coli and Aerobacter aerogenes, it turned out to be effective for the rapid identification of Candida albicans and was found useful for the identification of coagulase-positive Staphylococci.

REQUIREMENTS
COMPOSITION:
(Ingredients Grams/Litre) Pepton 10.0 Lactose 10.0 Dipotassiummonohydrogenphosphate 2.0 Methylene Blue 0.065 Eosine Y 0.4 Agar 15.0 Final pH 7.1 +/- 0.2 at 37C

MATERIALS USED:
Laminar air flow Conical flasks Petriplates Glass rods Autoclave Rotary shaker

PROCEDURE
Add all of the above mentioned components in given measurements to a conical flask.

Suspend the entire 37.5 g of mixture in in 500ml of distilled water. Dissolve the components. Make upto 1 litre by adding distilled water. Heat (if necessary) to dissolve completely. Sterilize by autoclaving at 121C for 15minutes. Cool to 60C. Shake the medium in order to oxidize the methylene blue and to suspend the precipitate. Pour into petriplates and allow to set.

EMB agar plate

Storage: Store prepared media below 8C and protected from direct light. Store dehydrated powder in a dry place, in tightlysealed containers at 2-25C. Appearance: Faintly violet to pink, homogeneous, hygroscopic powder. Gelling: Firm Color and Clarity: Deep red-brown, clear to slightly turbid

STEP 2

SAMPLING OF INTESTINAL MICROFLORA

In order to collect the intestinal microflora, the subject is to take a rectal swab kit to the restroom and obtain a sample. It is simply done by inserting a cotton swab into the rectum and making a sweep to obtain the sample on to the swab. Rectal Swab Kit This kit consists of a sterilized cotton swab in a test tube. As soon as you have obtained a sample, bring the swab back to the lab and plate it on different solid media. One cotton swab will have enough bacteria on it to inoculate several plate
Diagramatic representation on how to conduct a rectal swab

RECTAL SWAB PROCEDURE:

Wash hands and put on protective gloves. Gently insert the swab (provided with the kit) about 1.2 inches (3 centimeters) into rectum. Gently swirl the swab in a circular motion for 15-30 seconds. Remove cap from the tube containing the Rectal Swab Collection and Preservation Reagent. Slowly remove the swab from the patients rectum without touching the skin and insert into the tube.

Sterilzed Swab Stick

Image of a Swab Stick with container provided in the kit

Swirl the swab in the liquid media. Break off the end of the swab using scissors and recap the tube tightly. Discard the rest of the swab applicator in appropriate waste container. Use the provided labels to label the tube with the subjects ID and collection date. Place the tube into a provided zip-lock specimen bag. Remove gloves, wash hands thoroughly. Gloves should be discarded in appropriate waste container. Storage: Specimens should be held at room temperature until transport. Specimens may be held or shipped to the testing laboratory at ambient temperature for up to 7 days. Specimens held longer should be kept at -80C or lower until testing.

STEP 3

CULTURING OF INTESTINAL MICROFLORA

Once the sample is obtained, it is ready for culturing. A suitable media is prepared and allowed to set in a petriplate, and the sample is inoculated on to the media. After the incubation period, bacterial growth in form of colonies can be observed on the plate. In this case the are the bacteria that lines our intestines.

REQUIREMENTS
MATERIALS REQUIRED: LAF Burner EMB plate Inoculation loop Paraffin tape Incubator

PROCEDURE
The entire PROCEDURE is to be conducted in a sterilized LAF. Remove the top of the EMB plate. Keep it in your hand, and do not lay it on any non-sterile surface, or you may introduce bacteria onto it. Remove the swab from its protective cover. Gently swipe the swab back and forth on the surface of the agar plate. Take care not to press too hard, as the surface is delicate and may be damaged. Make a zig-zag pattern, and do not touch any part of the agar twice. Replace the top to the petri dish. Seal the plate with paraffin tape. Place the petri dish into an incubator set at approximately 37degrees Celsius (this is the temperature at which most bacteria thrive, and is the temperature of the normal human body). If you do not have an incubator, you can keep the Petri dish at room temperature, but it may take longer for the bacterial colonies to form.

 After 24 to 48 hours, remove the petri dish from the incubator and examine the growth of the bacterial colonies. It is safest to observe these bacteria through the top of the dish, as opening the dish can spread the bacteria (which are now higher in number) into the surrounding air.

EMB plate showing bacterial growth

STEP 4

ISOLATION OF E.COLI
Isolation of E.coli is the easiest step since we have used EMB agar as the media.

PRINCIPLE

EMB plate showing mettalic green coloured E.coli colonies

The presence of the colorants Eosine Y and Methylene Blue inhibits the growth of most of the common accompanying Gram-positive microorganisms. Levine described this classical method to identify E.coli from other coliforms as Aeorobacter aerogenes . Lactose is added as distinctive carbon and energy source. In combination with the added dyes it allows to distinguish between lactose-positive and lactose-negative organisms. Lactose positive cultures are generally dark violet (Enterobacter, Klebsiella, E.coli), while lactose negative organisms (Salmonella, Shigella) have only peptone as energy source are colourless. Some gram-positive bacteria, such as fecal streptococci, staphylococci will may grow on this medium as

inhibited small colonies. A number of nonpathogenic, lactosenonfermenting gram-negative bacteria will grow on this medium and must be distinguished from the pathogenic strains by additional biochemical tests. Cultural characteristics after 24-48 hours at 35C. However it is easy to identify Escherichia coli as it appears as 2-3mm, dark violet cultures with black center and green metallic shine

STEP 5

CULTURING OF E.COLI
Before proceeding to extraction of plasmid, we have to obtain a pure culture. Right now, the EMB agar plates contain a mixture of organisms. To obtain a pure culture, we will restreak a single E. coli colony on another agar plate. For this step, a new media can be chosen. Lb broth is the media chosen because it is the most common n easy medium to grow bacteria on. Luria-Bertani broth, also known as LB broth or LB medium, is the most common liquid medium used to grow bacteria such as E. coli. It was named after two scientists who created it in the 1950s while they were studying phages. LB broth is an excellent medium because it is very efficient at stimulating growth and is suitable for many different organisms. LB broth is categorized as a rich medium, meaning it contains all the nutrients such as peptides and peptones, vitamins, and trace elements needed for bacteria to proliferate. The recipe for the broth has changed little over the years and consists of three main ingredients: yeast extract, tryptone, and sodium chloride (salt). Yeast extract is basically a powdered form of the yeast found in the baking section of grocery stores. Tryptone is a pancreatic digest of the protein casein. Yeast extract and tryptone provide vitamins and amino acids, respectively, that the bacteria need to grow. Finally, sodium chloride is added to keep the broth at a certain ionic strength.

PREPARATION OF MEDIA
REQUIREMENTS
COMPONENTS:
10g Bacto-tryptone 5g yeast extract 10g NaCl Distilled H2O

 15g agar Final pH: 7.5

MATERIALS USED:
Laminar air flow Conical flasks Petriplates Glass rods Autoclave Rotary shaker

PROCEDURE
Dissolve 10 grams of tryptone, 5 grams of yeast extract, and 10 grams of sodium chloride in 1 liter of deionized water. Adjust the pH of the solution to 7.4 using sodium hydroxide. Add 15 grams of bacteriological agar to solidify the broth. Autoclave at 121C for 20 min to sterilize the broth. Let the mixture cool down to about 50 to 60C Pour the solution into petri dishes.

Pour plate technique

 The broth can be stored sealed at room temperature. To promote faster growth, the medium can be supplemented with 0.1% glucose or 0.4% glycerol.

LB plates
Storage: Plates can then be stored at 4C in plastic bags.
Appearance: a strong yellow color and an extremely pungent smell. Gelling: Firm Color and Clarity: yellow, clear.

 STREAKING OF E.COLI
MATERIALS REQUIRED
LAF Inoculation loops LB plates Flame Paraffin tape

PROCEDURE
Sterilize an inoculating loop by placing it at an angle over a flame. Remove the lid from a culture plate containing the desired microorganism. Cool the inoculating loop by stabbing it into the agar in a spot that does not contain a bacterial colony. Pick a colony and scrape off a little of the bacteria using the loop. Be sure to close the lid. Using a new agar plate, lift the lid just enough to insert the loop. Streak the loop containing the bacteria at the top end of the agar plate moving in a zig-zag horizontal pattern until 1/3 of the plate is covered. Sterilize the loop again in the flame and cool it at the edge of the agar away from the bacteria in the plate that you just streaked. Rotate the plate about 60 degrees and spread the bacteria from the first streak into a second area using the same motion in step 6. Sterilize the loop again using the procedure in step 7. Rotate the plate about 60 degrees and spread the bacteria from the second streak into a new area in the same pattern. Sterilize the loop again. Replace the lid and invert the plate. Incubate the plate over night at 37 degrees Celsius.

You should see bacterial cells growing in streaks and in isolated areas .

E.coli bacteria growth seen on LB agar plate

CONCLUSION
The culture o e.coli obtained can be further studied to identify the disease causing strains. Mostly, e.coli are friendly probiotics but certain strains of it can cause diseases that could be fatal if not diagnosed n treated immediately. So necessary precautions need to be taken. Most of the infection are caused by the E. coli serotype O157:H7. E. coli O157:H7 is markedly different from other pathogenic E. coli, as well. In particular, the O157:H7 serotype is negative for invasiveness (sereny test), adheres through the E. coli common pilus (ECP), and doesn't produce heat stable or heat labile toxins. In addition, E. coli O157:H7 is usually sorbitol negative whereas 93% of all E. coli ferment sorbitol. E. coli O157:H7 also lacks the ability to hydrolyze 4-methylumbelliferyl--D-glucuronide (MUG) and does not grow at 45 C in the presence of 0.15% bile salts. Because of the latter characteristic this serotype cannot be isolated by using standard fecal coliform methods that include incubation at 45 C. While relatively uncommon, E. coli O157:H7 can naturally be found in the intestinal contents of some cattle. Because ruminants lack a receptor for the bacteria, it does not cause disease in them and is considered commensal.

REFERENCE
Escherichia Coli Infection -Shannon D Manning Microcosm: E. coli and the New Science of Life -Carl Zimmer Manual of clinical microbiology, Volume 1 -Patrick R. Murray, Ellen Jo Baron, American Society for Microbiology Bacterial Culture Media -Sridhar Roa PN

http://en.wikipedia.org/wiki/Escherichia_coli http://www.about-ecoli.com/ http://www.ecolirep.umn.edu/ecoliisolation.shtml http://lib.bioinfo.pl/meid:1005