What is gram staining?

Indications and Procedures

The observation and identification of bacteria are of obvious primary importance in the study of microorganisms. Even with the use of powerful microscopes, direct observation of unstained bacteria is difficult. The use of stains to increase their contrast with the background allows bacteria to be observed more easily.

As a result of resident acidic groups—polysaccharides or nucleic acids—the surfaces of bacteria tend to be negatively charged. Conversely, the dye portion of common stains such as methylene blue or crystal violet consists of positively charged ions. For staining purposes, a sample of bacteria is placed on a glass slide and allowed to dry. The solution of stain is flooded over the bacterial “smear” for about a minute, and the slide is then rinsed. The main purpose of such simple stains is to allow the cells to be observed.

In contrast with simple stains, differential staining methods do not stain all cells in the same manner. Bacteria grown under different environmental conditions, or bacteria that may differ from one another in their physical structure, will exhibit different staining properties when treated with differential stains. Gram staining (also called Gram’s stain) is an example of a differential stain.

Gram staining is a relatively simple procedure and is among the first practices learned by students in microbiology laboratories. The process begins with the preparation of a bacterial smear on the slide. A stain, crystal violet, is allowed to flood the dried smear. The slide is rinsed, and a solution of iodine is dropped over the smear. The iodine functions as a mordant, fixing the crystal violet into a complex insoluble in water. Following another rinse, the smear is covered with either an alcohol or an acetone “destaining” solution for several seconds, rinsed again, and counterstained with the red dye safranin. After a last wash, the bacteria are observed with a microscope. If they were not destained by the alcohol step, retaining the blue or violet color, they are considered gram-positive; if they have stained pink because of the counterstain safranin, they are considered gram-negative.

The precise means by which gram staining works is not entirely clear. The cell wall structure of gram-positive bacteria
either prevents the alcohol/acetone solution from removing the crystal violet-iodine complex from the cell or prevents the solution from having access to the complex. Though the question remains whether the cell wall structure is the sole determining factor in the differential procedure, there is no doubt that the cell wall features are primary factors in the determination of gram-staining results. Therefore, the structure of the cell wall in most bacteria reflects the gram-staining characteristics.

The cell wall structure found in gram-positive bacteria differs significantly from that in gram-negative cells. While both contain a rigid layer called the peptidoglycan, the peptidoglycan layer is much thicker and makes up a significantly larger portion of the cell wall in gram-positive bacteria. In contrast, a significant portion of the cell wall found in gram-negative bacteria is composed of lipid derivatives.

The peptidoglycan portion of the cell is composed of repeating units of two sugar derivatives: N-acetylglucosamine and N-acetylmuramic acid. The peptidoglycan within the wall is in the form of sheets, layered on top of one another. In gram-positive bacteria, approximately 90 percent of the cell wall material consists of peptidoglycan; among gram-negative bacteria, about 10 percent of the wall is represented by this rigid layer.

These cell wall structures are stabilized by short chains of amino acids that cross-link the layers of peptidoglycan. Formation of the cross bridges is an enzymatic process called transpeptidation. The antibiotic penicillin inhibits the enzyme that carries out the formation of such cross-links. The result is a weakening in the cell wall, and possibly cell death. Since the peptidoglycan layer of gram-negative bacteria represents a much smaller proportion of the cell wall, such microorganisms are often more resistant to the action of penicillin than are gram-positive bacteria.

During the gram-staining procedure, decolorization of the cell is carried out during the wash with alcohol or acetone. The thick peptidoglycan layer found in gram-positive bacteria, however, prevents movement of the crystal violet-iodine complex from the cell. Thus, the cells do not decolorize; they retain their violet appearance.

The peptidoglycan layer is a small proportion of the gram-negative cell wall. Much of the outer wall in these bacteria is a layer of lipopolysaccharide (LPS), which acts as a physical barrier but also contains pharmacological properties. The LPS layer is a complex structure containing a lipid portion (lipid A), a core polysaccharide consisting of a variety of sugars, and an outer layer of branched sugars called the O-region (O-polysaccharide). The LPS layer is anchored to the thin peptidoglycan portion of the cell wall through a lipoprotein complex. The LPS portion of gram-negative cell walls is often termed the endotoxin because of its pharmacological activity. Release of LPS as a result of cell death during certain types of infection can result in high fever or shock.

Since the cell wall of gram-negative bacteria contains proportionately little peptidoglycan, the crystal violet-iodine complex is easily removed during the gram-staining procedure. Following the alcohol step, the cells again appear colorless. Therefore, when they are counterstained with the safranin, the bacteria will appear pink.

An evaluation of gram-staining characteristics is generally the first step in the identification of newly isolated bacteria. Most bacteria can be classified as either gram-positive or gram-negative, and this step, along with characterization of the shape of the organism, is of immense importance in narrowing down the possible identities of an isolate.

Further means of identification generally involve the use of selective or differential types of media. These processes use the biochemical properties of bacteria for their identification. A selective medium is one in which chemical compounds have been added that inhibit the growth of certain forms of bacteria but allow the growth of others. For example, the chemical dye eosin-methylene blue (EMB) inhibits the growth of gram-positive bacteria while allowing gram-negative bacteria to grow. If a mixed culture of bacteria is inoculated onto EMB medium, only the gram-negative microorganisms will grow. A differential medium will allow a variety of bacteria to grow, but different types of bacteria may produce different reactions on the medium. Since EMB agar contains lactose as a carbon source, it is also a differential medium. Bacteria that ferment lactose produce a green metallic color of colony on EMB; bacteria that do not ferment lactose produce a pink colony.

Biochemical tests are more useful for the identification of organisms that are gram-negative than for those that are gram-positive. Biochemical variations among both genera and species of gram-positive bacteria tend to be too variable for effective identification of these organisms. By contrast, such biochemical results among gram-negative bacteria generally do not vary significantly within the species and hence are useful means of further identification.

The biochemical tests used for identification of gram-negative bacteria can be summarized in the form of a flowchart. Such charts represent the series of tests that divide bacteria into smaller and smaller groups. For example, following a determination of morphological and gram-staining characteristics, differential tests may be carried out to observe the ability of the bacteria to ferment various types of carbohydrates. A series of broths containing such sugars as glucose, lactose, or sucrose are inoculated. Generally, a pH indicator such as the chemical phenol red is included, as is an inverted glass tube (Durham tube) for observation of gas production. If the organism can ferment lactose and produces acid and gas, the broth tube of lactose will appear yellow and there will be a gas bubble in the Durham tube. If the organism does not ferment lactose, no growth or change from the red color of the broth will be observed in that tube.

Further differentiation of either lactose-positive or lactose-negative organisms, to continue with this particular example, can be carried out with other biochemical tests. Certain species of bacteria are capable of removing a molecule of carbon dioxide from amino acids; others are not. In some cases, multiple tests can be run at the same time. For example, a common differential test for gram-negative bacteria uses a medium called triple sugar iron (TSI) agar. TSI agar contains a small amount of glucose and larger amounts of lactose and sucrose, hence the designation of triple sugar. Iron is also contained in the medium. The agar is prepared in a test tube and allowed to harden on a slant. Organisms are inoculated onto the surface of the slant and stabbed into the butt of the slant. If glucose alone is fermented, only enough acid is produced to turn the butt yellow. If either lactose or sucrose is fermented, both the slant and butt of the agar will turn yellow. Production of hydrogen sulfide gas is indicated by a black precipitate from iron sulfide; other gas production is indicated by bubble formation in the region of the stab. In this manner, inoculation of a single type of medium can provide multiple tests for identification.

At one time, each of these differential tests had to be carried out individually. Beginning in the 1970s, however, a variety of media kits became available that allow fifteen to twenty tests to be run simultaneously. These kits consist of strips of miniaturized versions of biochemical tests that permit the rapid identification of gram-negative bacteria.

Even though some biochemical tests are less helpful in the identification of gram-positive bacteria, some characteristics of these organisms can be used. These organisms may be round (cocci) or rod-shaped (bacilli). If bacilli, they may be aerobic (they utilize oxygen) or anaerobic (they do not utilize oxygen). By testing for coagulase, an enzyme that will cause the coagulation of plasma, cocci can be further differentiated.

Finally, serological methods can be used in the identification of either gram-positive or gram-negative organisms. In these tests, a fluorescent dye is attached to molecules of antibodies, proteins directed against the surface molecules of specific bacteria. The ability of the antibodies to attach to bacteria is indicative of the species.

Uses and Complications

The use of gram-staining methodology is arguably the single most important step in the identification of microorganisms; its applications are far-ranging. Most diseases of humans and other animals, as well as of plants, are caused by microorganisms. Isolation and identification of disease-causing bacteria are key aspects in understanding the etiology of such diseases. Many aspects of technology, from the discovery or development of new antibiotics to the development of new strains of microbes, utilize such methodologies as gram’s stain in the identification of fresh isolates.

Clinical methods for the identification of infectious agents follow a series of defined steps. The particular material involved depends on the type and site of infection and can include such fluids as blood, urine, pus, or saliva. The specific symptoms of the illness may also provide clues as to the particular agents involved.

For example, among the most common infections are those of the urinary tract. These are particularly common nosocomial, or hospital-acquired, infections. A clean-voided or catheter-collected urine specimen is inoculated onto a plate containing selective or differential agar media using a calibrated loop. The number of bacterial colonies that grow after incubation can then be counted to estimate the concentration of bacteria present in the urine. This helps determine if an infection is present. Generally speaking, such infections are usually associated with gram-negative bacteria. The majority of these infections, about 90 percent, are caused by
Escherichia coli
(E. coli), a common intestinal organism. To a lesser extent, such infections may be associated with other genera such as
Klebsiella
,
Pseudomonas
, Proteus, or
Streptococcus
. All but Streptococcus are gram-negative bacilli.

Confirmation of the gram morphology follows growth on selective media. The media of choice in this example are those selective for gram-negative bacteria: either eosin-methylene blue or MacConkey agars. Both inhibit the replication of bacteria such as
Staphylococcus
, commonly found on the surface of the skin and a possible contaminant during the swabbing of the site of infection.

The presence of the sugar lactose in either MacConkey or EMB agar allows these media to be differential in addition to being selective. Lactose fermenters such as Escherichia, Klebsiella, or
Enterobacter
will produce pink colonies on MacConkey agar, while gram-negative organisms such as Proteus, Pseudomonas, or
Salmonella
, which do not ferment lactose, will produce colorless colonies on this medium. Analogous results can be seen with other differential enteric agars. More detailed types of analysis using other forms of media or utilizing immunological methods may be necessary to fine-tune the diagnosis, or antibiotic susceptibility tests may simply be conducted to determine the treatment of choice.

In some instances, gram morphology may be sufficient for the identification of a microorganism. For example, the presence of gram-negative cocci in a cervical smear from a patient suspected of having contracted a sexually transmitted disease is indicative of a
Neisseria gonorrhea infection. The identification can be confirmed using immunological methods or through growth on selective media such as Thayer-Martin agar, which contains antibiotics inhibitory to most other gram-negative bacteria.

If the clinical sample consists of blood or cerebrospinal fluid, both of which are normally sterile, either gram-negative or gram-positive organisms may be involved. The initial step toward identification is a gram stain of the material. Gram-negative bacteria can be identified using methods already described. Generally speaking, the bacterial content of blood during bacteremia will be too low for ready observation. For this reason, blood samples are inoculated into bottles of nonselective growth media, one of which is grown under aerobic conditions and one under anaerobic conditions. If and when growth becomes apparent, smears are prepared for gram staining.

Gram-positive cocci will almost always be members of either of two genera: Staphylococcus or Streptococcus. The two can be differentiated on the basis of catalase production, an enzyme which degrades hydrogen peroxide; staphylococci produce the enzyme, while streptococci do not. A variety of commercial kits are available for rapid identification of species. These contain a battery of tests based on biochemical properties of the organisms, including tolerance of high salt, fermentation of unusual sugars, and growth characteristics on blood agar plates (nutrient agar containing sheep red blood cells).

The identification of gram-positive bacilli is more difficult, given their biochemical variation even within the genus. The major subdivisions of this group are based on their tolerance of oxygen. Obligate gram-positive anaerobes, organisms that cannot tolerate oxygen, include the genus
Clostridium
, members of which cause tetanus, gangrene, and food poisoning. Aerobes and facultative anaerobes, which are oxygen-tolerant, include the genera Bacillus,
Corynebacterium
, and
Listeria
. Further identification often requires immunological means.

The gram-negative bacillus E. coli is frequently used as a marker for sewage contamination of water supplies. Since it is a common intestinal organism and rarely found in soil, its presence in water samples is indicative of possible fecal contamination of that water. Testing for the presence and level of E. coli utilizes the biochemical properties of the microbe. Various quantities of the water sample are placed in tubes of lactose broth; growth is indicative of a lactose fermenter and is presumptive for the presence of E. coli. A sample of the lactose culture is then streaked on EMB agar. The development of metallic green-colored colonies of gram-negative bacilli confirms the presence of E. coli. The smaller the volume of the water sample that produced growth in lactose, the higher the level of E. coli in that sample. In a sense, E. coli serves as a surrogate marker in these tests. It may not itself be a pathogen (though some strains of E. coli may indeed cause severe intestinal infections), but other gram-negative intestinal pathogens such as Salmonella,
Shigella
, or
Vibrio
, even if present in water supplies, may be in a concentration too low for ready detection. Therefore, the presence of E. coli suggests possible fecal contamination, allowing for proper sewage treatment. Conversely, the absence of E. coli in the water sample indicates that fecal contamination, and therefore the presence of other intestinal pathogens, is unlikely.

Perspective and Prospects

During the latter portion of the nineteenth century, the role of bacteria as etiological agents of disease became apparent. Eventually the experimental observations linking the presence of bacteria with various illnesses coalesced in the so-called germ theory of disease. During the early 1880s, the German physician Robert Koch, along with his colleagues and students, developed an experimental method that could be applied to associate a particular organism with a specific disease. These procedures eventually became known as Koch’s postulates. Inherent in Koch’s postulates was the necessity to observe the microbial agent, either in tissue or following growth in the laboratory. Staining methods, however, were often crude or imprecise. The best one might hope for was to be able to at least observe the organism.

Hans Christian Gram, a Danish physician working with Carl Friedlander in Berlin during the early 1880s, was able to introduce a highly effective method of staining bacteria. Gram’s method was a modification of that developed earlier by Paul Ehrlich. The procedure began by first staining the sample with Gentian Violet in aniline water, followed by treatment with iodine in a potassium iodide solution. Gram found that when tissue sections or smears treated in such a manner were washed with dilute alcohol, certain types of bacteria (or schizomycetes, as they were then known) became decolorized (gram-negative), while other forms of bacteria retained their violet appearance (gram-positive). The procedure, published in 1884, was shown to be applicable for most types of bacteria. As a result, a process for differentiation between various types of bacteria became available. In addition, the ability to detect smaller quantities of bacteria in tissue increased significantly.

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