Isolation of Escherichia coli

Many different types of bacteria reside within our mouths. An even greater number of bacteria reside within our intestines. In fact, it has been estimated that the average adult harbors 100 trillion microorganisms on and in his or her body. Today, we will begin to examine some of the normal microflora of our intestines and each person will isolate his/her very own Escherichia coli.

Day 1

In order to collect your intestinal microflora, everyone should take a rectal swab "kit" to the restroom and obtain a sample. This kit consists of a sterilized cotton swab. 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 both plates.

First, streak the cotton swab on a tryptic soy agar plate. Next, rotate the cotton swab slightly and streak it on an EMB plate. Dispose of cotton swabs in an autoclave bag. Label all plates and incubate them overnight at 37oC.

Day 2

Observation of colonies

Remove your plates from the incubator. Visually examine your plates and the plates of several lab-mates. While examining these plates, consider the following questions. What differences do you notice in bacterial growth on the tryptic soy agar plate and the EMB plate? What factors could account for these differences? Do your intestinal microflora seem to differ (based on colony morphologies) from those of your fellow lab-mates? What factors could account for these differences?

Many bacterial species produce very distinctive colonies on EMB agar. As we observed previously, E. coli colonies are very dark, with a metallic green sheen, P. aeruginosa colonies are colorless, and Enterobacter aerogenes colonies are pink with darkened centers. Everyone should be able to identify E. coli colonies on EMB agar plates.

While selective and differential media often are useful in tentatively identifying some microorganisms, additional methods usually are needed to positively identify a species. As we have seen, a series of biochemical tests generally are employed.

Restreaking for isolation

Before proceeding with our characterization of E. coli, we have to obtain a pure culture. Right now, your EMB agar plates contain a mixture of organisms. To obtain a pure culture, we will restreak a single E. coli colony on another EMB agar plate. Touch the needle end of a sterile loop to a well isolated E. coli colony. Streak the needle across a third of the new EMB plate. Using a new, sterile needle, streak for isolation as we have done in the past. Incubate the plate at 37oC.

After 18-24 hours, remove the plate from the incubator and examine it. Unlike the colonies on the first EMB plate, all of the colonies on this plate should look the same. All these colonies are clonal populations of your E. coli isolate. We will save (in the refrigerator) this plate and use it as our source of bacteria throughout the semester.

Repeat this procedure with a TSA plate to isolate an unknown species.


Perform whatever tests (if any) that you feel are necessary to confirm that you have isolated E. coli and to make a preliminary identification of your unknown isolate.

Night before March 10 or 11 lab session

During the lab session on March 10 or 11, each person will need an overnight culture of his/her E. coli. Therefore, each person must start an overnight culture the night before lab. Using a sterile needle, remove a colony from your plate and use it to inoculate a tube of broth. Label it and place it in the 37oC shaker at an angle. Remember to leave the cap of the tube slightly loose. Return the agar plates to the refrigerator.
Normal Mouth Microflora (+ or - refers to Gram stain properties)
Streptococcus salivarius +, form short chains, alpha or non-hemolytic
Streptococcus pneumoniae +, form short chains, alpha hemolytic
Streptococcus mitis +, form short chains, generally non-hemolytic
Streptococcus pyogenes +, form short chains, beta-hemolytic
Staphylococcus epidermidis +, form tetrads, chains, or clusters, gray/white colonies, Generally non-hemolytic
Staphylococcus aureus +, form tetrads, chains, or clusters, gray to deep yellow colonies, various degrees of hemolysis
Lactobacillus species +, rod-shaped
Klebsiella species -, rod-shaped, mucoid colonies, ferment lactose
Fusobacterium nucleatum -, rod-shaped

Normal Intestinal Microflora  
Staphylococcus epidermidis see above
Staphylococcus aureus see above
Streptococcus mitis see above
Lactobacillus species see above
Clostridium species +, rod-shaped, anaerobes, often beta-hemolytic
Escherichia coli -, rod-shaped, ferment lactose
Klebsiella species see above
Pseudomonas aeruginosa -, rod-shaped, occasionally occurs in pairs or short chains, produce various pigments which result in colonies of different colors, do not ferment lactose
Bacteroides species -, rod-shaped or coccobacilli, anaerobes