Bacterial Identification

To learn more about how to identify bacteria, we will plate a series bacterial species on several different selective and/or differential media.


Unknowns A through J, sewage isolates ?1, ?2, ?3


Tryptic soy agar

Eosin methylene blue agar

Mannitol salt agar

MacConkey agar

Phenylethanol agar

Triple sugar iron slants

Plate two bacterial unknowns (A - J) per plate on each of the available media. Incubate at 37oC overnight.

Observing Plates

When observing your plates, you should notice several things. First, in locations where bacteria are crowded closely together, confluent growth appears. In areas where the bacteria are more spread out, though, individual colonies should appear. Each colony is the result of a single bacterial cell undergoing multiple divisions during the period of incubation. Each colony, therefore, represents a clonal population of bacteria. If your plate streaking was done correctly, the initial streaking sector should contain a fairly confluent lawn of bacteria. The second or third sectors should contain individual colonies.

Growth characteristics

As you observe your plates, you also should notice that the colonies of the different bacterial species have different appearances. Furthermore, the colony morphology of a given bacterial species is fairly consistent. As a result, colony appearance often can be used by microbiologists as a means of identifying bacteria.

When examining colony morphology, consider the following traits:


Whole colony shape (circular, irregular, rhizoid)

Colony edge (smooth, filamentous, undulating)

Elevation (flat, raised, convex, crateriform)

Surface (wrinkled, rough, waxy, glistening)

Opacity (transparent, translucent, opaque)


Color (red, yellow, white)

Water solubility (water soluble - color tints surrounding media)


In addition to observing the colonies, compare the attributes of colonies observed on the different types of media. Think about the following questions:

What is a selective medium?

What is a differential medium?

How does EMB medium differ from MacConkey medium?

Were any of these media useful in identifying the bacteria?

How would you change the experiment to more conclusively identify the bacteria?

Be sure you understand why each of the media produced the observed results.

Basic microscopy

Obviously, the use of a microscope is a very important skill for a microbiologist. In this course, we will be using compound light microscopes.  A tutorial on microscope use is available.

In compound microscopes, two or more sets of lenses are used. Total magnification is calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens. Thus, with our scopes, magnification varies from 100x to 1000x. To view bacteria, we will use the 40x and 100x objective lenses primarily. In addition to magnification, the resolving power (ability to distinguish two adjacent objects) of a microscope is vitally important.

A few basics about the use of microscopes:

Generally, objects first should be examined with the low power objective. Once the material is in focus, you can switch to higher powered objective lenses.

Our microscopes are parfocal, that is, once a sample is brought into focus with a low powered objective lens, it will remain in focus when you switch to a higher powered lens. Therefore, there should be no need to use the coarse focus knob when switching to another objective.

These microscopes are fairly expensive and, to a degree, fairly fragile. As a result, be careful when handling the scopes. Carry a scope with one hand in the handhold in the back of the scope and one hand underneath the scope. Don't set them too close to the edge of the bench. Keep electrical cords out of the way.

Despite the cost and fragility of the scopes, don't be afraid to use them. Try out the various buttons, switches, and knobs. Investigate what settings allow you to see your sample best. These microscopes are here for your use.

Gram staining

Staining is an integral method of identifying microorganisms. Basically, stains, or dyes, bind to a cellular component and give it color. As a result, the organism is visible against an otherwise uncolored background. Stains can serve two functions. First, a simple stain can be used to determine shapes, sizes, and arrangements of cells. Such a stain reacts similarly with all cells. In other words, it does not provide a means of differentiating various types of microorganisms. A differential stain, on the other hand, usually employs two or more stains that differentially stain different microorganisms. Thus, one can use a differential stain to determine what types of microorganisms are present in a mixture or to identify various cellular components. Most bacterial stains are cationic. Because bacteria generally possess negatively-charged cell membranes, positively-charged (cationic) stains will bind readily to the membrane.

Today, we will use the most common differential staining technique in microbiology the Gram stain. In this procedure, Gram-positive and Gram-negative bacteria are stained initially with crystal violet, causing both types of bacteria to appear purple. Iodine then is added to the sample to form an iodine-crystal violet complex in the cell wall. The decolorizer (usually ethanol or an ethanol/acetone mixture) removes most of this complex from Gram-negative cells, thereby removing the purple color, and also increases the porosity of these cells. Finally, safranin counterstain is added to the sample. This stain permeates the decolorized Gram-negative bacteria, giving them a pink color. Today, we will prepare slides of and Gram stain samples of unknown bacteria and our own oral bacteria.

Sample Preparation

Bacterial colony growing on solid media:

Place drop of water on clean slide
Isolate bacterial colony with sterile loop
Suspend bacteria in water. Spread out on slide
Allow to air dry
Heat fix cells by passing through flame 3-4 times

Bacteria growing in liquid media

Obtain drop of bacterial suspension with a sterile loop
Spread suspension on clean slide
Allow to air dry
Heat fix cells

Oral bacteria

Swab inner cheek with cotton swab
Smear specimen on clean slide
Allow smear to air dry
Heat fix cells
Note: Heat fixation is an important step of this procedure, serving three main purposes.
Heat fixation:
Kills organisms
Causes cells to adhere to slide
Alters cells so they more readily accept dyes
If a sample is under-heated, cells will not adhere well to the slide and may wash off during the process. If a sample is over-heated, cells may be incinerated, thereby destroying the cells. Finally, if a sample is not air dried prior to heat fixation, the heating may cause the liquid to boil, again destroying the cells.

Once you have prepared your slides, proceed with the actual staining procedure.

Gram staining

Dunk slide in crystal violet. Remove and allow to stand for one minute.
Rinse gently with water.
Dunk slide in Gram iodine. Remove and allow to stand for one minute.
Rinse gently with water.
Dunk slide in decolorizer for approximately 10 seconds.
Rinse gently with water.
Dunk the slide in safranin. Remove and allow to stand for one minute.
Rinse gently with water.
Blot dry and observe with microscope.

Note: for best results, these slides should be viewed with the 100x oil immersion lens.

Take note of the different staining properties, morphologies, and arrangements you observe with the pure cultures and with your own oral swab. What unusual things do you see in the oral swab? How useful is the Gram stain in identifying microorganisms? How useful do you think the Gram stain is in making a diagnosis in a medical setting?

Triple sugar iron medium

The large intestine of humans contains a large number of microorganisms.  There are estimates, in fact, that human fecal material normally contains more than 1011 bacteria per gram.  Most of these organisms are non-pathogenic and some even benefit us by, for instance, manufacturing vitamin K.  The most common genera present in the human large intestine include Bacteriodes, Bifidobacterium, Enterococcus, Lactobacillus, Escherichia, Enterobacter, Citrobacter, and Proteus.   As a whole, these bacteria often are referred to as enterics.  More appropriately, the enterics are bacteria in the family Enterobacteriaceae (Gram negative, rod-shaped, facultative anaerobes).

While most microorganisms found in the large intestine are non-pathogenic, a number of gastrointestinal diseases are caused by bacteria.  Most often, these bacteria are transmitted via ingestion of contaminated food or water, a transmission mechanism referred to as fecal-oral spread.  Because of these pathogens, several media have been developed to differentiate the intestinal bacteria.  MacConkey medium differentiates lactose fermenters from lactose non-fermenters.  Gram negative, rod-shaped lactose fermenters are referred to as coliform bacteria and, generally, are non-pathogenic.  Triple sugar iron medium often is used to further characterize intestinal microflora.  This medium contains:

0.1% glucose

1.0% lactose

1.0% sucrose

0.02% ferrous sulfate

phenol red

nutrient agar

The phenol red is a pH indicator.  If the medium becomes acidic, then the phenol red turns yellow.  If the medium becomes alkaline, then the phenol red turns purple.  If an organism can only ferment glucose, then the medium initially will turn yellow.  Because there is so little glucose in the medium, however, the bacteria quickly will exhaust the glucose supply and begin to oxidize amino acids for energy.  The oxidation of amino acids produces ammonia as a by-product.  The ammonia will cause an increase in pH and a return to a red or purple color on the surface of the slant.  Therefore, organisms that can only ferment glucose will produce a slant with a red surface and yellow butt.  If the organism being tested can ferment lactose and sucrose, then the entire tube will turn, and remain, yellow.  As we discussed in class, many metabolic reactions result in the production of gas as a by-product.  The production of gas can be detected in TSI slants by the presence of bubbles within the agar.  Furthermore, if the produced gas is H2S, it will react with the ferrous sulfate to produce ferrous sulfide, a black precipitate.

Each group will receive five slants of TSI medium.  One tube will be a negative control.  The other four slants will be used to investigate the properties Gram (-) rod-shaped unknown bacteria.

For each of four unknowns that appear to be Gram (-) rods, carry out the following procedure. With a sterile needle, pick an isolated colony from the TSA plate.  Stab the needle to the bottom of the slant, then streak the surface of the slant.  Incubate overnight at 37 degrees C and record your results.