Gram Stain

Protocol for Gram staining

Solutions:

Hucker's crystal violet reagent is made by mixing two solutions:

 Solution A:   Solution B:  

Crystal violet 2.0g Ammoniumoxalate 0.8g

Ethanol (95%) 20.0ml Distilled water 80.0ml

This mixture is stable and can be kept at room temperature for months. An iodine solution and counterstain are also required:

Stabilised Lugol- PVP complex:

  Counterstain:  

Iodine 1.3g Safranin 0.25g

KI 2.0g Ethanol (95%) 10.0ml

Polyvinylpyrrolidone 1.0g Distilled water 100.0ml

Add distilled water to 100ml      

Procedure:

Prepare a light suspension of cells from very young cultures grown on appropriate agar medium. If the suspension prepared is too turbid, dilute with distilled water.

1.       Add one drop to a clean glass slide and spread the drop with a loop over the surface of the slide. Allow to air-dry.

2.       Carefully fixate the cells by moving the slide into a flame (bacteria upwards).

3.       Flood the slide for 1 minute with Hucker's reagent.

4.       Wash the slide by dipping the slide into slow running tap water.

5.       Flood the slide with iodine solution for 1 minute.

6.       Place slide diagonally in glass box and rinse of iodine solution with safranin. 

7.       Add excess amount of fresh safranin and wait for 35 seconds.

8.       Rinse slide with water as described under (5).

9.       Allow the slide to air-dry.

10.   Examine the preparations with the oil immersion objective of the bright field microscope (do not use the phase-contrast objective!). A drop of oil can be placed on the slide directly.

11.   Gram-positive cells appear purple and Gram-negative cells pink. (The iris of the microscope condenser should be opened as wide as possible. With a closed condenser colours can hardly be discriminated.).

GRAM Micro-Organisms Staining Protocol

Prepared byROY ELLIS

IMVS Division of PathologyThe Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011

PrincipleThe cationic dye crystal violet is used to stain the nucleic acids of the micro-organisms and background tissues.  The crystal violet staining is then laked with iodine, forming a black complex.   Certain micro-organisms resist differentiation due to the impermeability of their cell walls.  However, using a suitable differentiator, (eg alcohol, aniline, or acetone), the tissue background and certain species of micro-organisms lose their staining, but take up a cationic dye of contrasting colour (usually red) subsequently applied. The blue-black staining micro-organisms are termed "Gram positive", whereas the micro-organisms that take up the counterstain (red) are termed "Gram negative".                         Gram positive micro-organisms           Gram negative micro-organisms                         Staphylococcus spp                              Coli-typhoid-dysentry group                        Streptococcus spp                               Gonococcus spp                        Pneumococcus spp                               Meningococcus spp                        B Diphtheria                                        Pasteurella pestis                        Chlostridium spp                                 Brucella spp                        B Anthracis                                         Salmonella spp                        Lactobacillus spp                                 Vibrio choleraTechnical Points 1.   A known positive control section must be used to ensure correct differentiation has

been achieved.2.   Step 6 - Differentiation with acetone is very rapid being only one or two seconds.  

The acetone should be poured liberally over the slide to ensure even decolourisation. Method

 1.   Bring sections to distilled water2.   On a rack, flood with filtered crystal violet   10 sec3.   Wash briefly in water to remove excess crystal violet4.   Flood with Gram’s iodine 10 sec5.   Wash briefly in water, do not let the section dry out. 6.   Decolourise with acetone until the moving dye front has passed the lower edge of the

section7.   Wash immediately in tap water8.   Note: If the section appears too blue repeat steps 6 and 79.   Counterstain with safranin 15 sec10. Dehydrate absolute alcohol, clear and mount. Results 

Gram positive micro-organisms............................... blue/black          Filaments of nocardia and actinomyces mycelia............ blue

Gram negative micro-organisms, Actinomyces clubs....... red          Nuclei .............................................................red

Reagent Formulae1.  Crystal violet stain

crystal violet (CI 42555 ) 2.0 g95% alcohol 20.0 mlammonium oxalate 0.8 gdistilled water 80.0 mlThe solution is prepared by dissolving the dye in the alcohol, the ammonium oxalate in the distilled water, and mixing the two solutions together.  The mixture is stable for two to three years.

 2.  Gram's iodine

 iodine crystals 1.0 gpotassium iodide 2.0 gdistilled water  300.0 mlDissolve the potassium iodide in 2 mls to 3 mls only of the distilled water - the crystals will dissolve and the solution will become very cold.  Dissolve the iodine crystals in the concentrated potassium iodide solution.  Dilute the mixture with the remainder of the distilled water. 

3.      Acetone     CARE - Fire hazard

 4.      Safranin References Gram C,(1884),Fortschr.Med.,2,185 Lillie RD,(1928),Archs.Path.,

 Lillie RD,(1977), The Gram Stain. A quick method for staining gram positive organisms in tissue. Arch Path., V5,p828-834.

The Test Sample

What is being tested?

A Gram stain is a laboratory procedure used to detect the presence of microorganisms, especially bacteria, in a sample taken from the site of a suspected infection. It gives relatively quick results as to the general type of bacteria that may be present. The Gram stain involves applying a sample from the infected area onto a glass slide and allowing it to dry. The slide is then treated with a special stain and examined under a microscope by a trained laboratorian. Any bacteria that may be present are categorized by color and shape during the microscopic evaluation:

Color — typically bacteria may be either "Gram positive" (purple) or "Gram negative" (pink)

Shape — the most common shapes include round (cocci) or rod-shaped (bacilli)

Additional information may be obtained by observing the groupings of the bacteria on the slide, such as cocci that are present singly, in pairs, in groups of four, in clusters or in chains, or bacilli that are thick, thin, short, long, or have enlarged spores on one end. Any bacteria that are present within the patient's white blood cell (intracellular) are also noted. The Gram stain color and the bacterial shape give clues as to what microorganism might be causing the infection. Examples of gram-positive cocci include Staphylococcus aureus, the bacterium associated with staph infections. An example of gram-negative bacterium is Escherichia coli, the cause of many urinary tract infections. Fungi (in the form of yeasts or molds) can also be initially identified with the Gram stain, but viruses cannot be seen with a Gram stain.

How is the sample collected for testing?

Several different types of samples may be collected for Gram stains. Some samples are collected using sterile swabs to obtain cells or exudate at the site of suspected infection.

Other samples, such as urine or sputum, may be collected in a sterile container. Some body fluids may be collected by needle and syringe. A swab may be used to collect a sample of bacteria grown and isolated in a culture.

NOTE: If undergoing medical tests makes you or someone you care for anxious, embarrassed, or even difficult to manage, you might consider reading one or more of the following articles: Coping with Test Pain, Discomfort, and Anxiety, Tips on Blood Testing, Tips to Help Children through Their Medical Tests, and Tips to Help the Elderly through Their Medical Tests.

Another article, Follow That Sample, provides a glimpse at the collection and processing of a blood sample and throat culture.

Is any test preparation needed to ensure the quality of the sample?

No test preparation is needed.

Gram stain preparation

Jim Hutchinson | 23 Jul 2010

Smears for Gram staining may be prepared from a variety of clinical specimens, broth cultures, or from colonies grown on solid media.

Always label the glass slide with appropriate information before applying material.

Specifics of preparation

Specimens received on swabsRoll the swab and dab the tip on the central area of the slide. It is important that you do this gently to avoid cellular destruction and disruption of bacterial arrangements.

Specimens not received on swabs: aspirates, exudates, sputa or any fluid too purulent or viscous for centrifugationIf the sample is sputa or pus – select portions which are purulent or blood tinged. Use a loop to spread the selected portion onto the slide to create a thin film. If the sample is extremely purulent or mucoid dilute the sample on the slide with a drop of sterile saline.

CSF and other thin body fluids requiring centrifugation prior to processing

1. When sample received in sterile container – aseptically pour off portion into properly labelled sterile screw-capped test tube – centrifuge at 3000 rpm for 10 minutes.

2. Apply a specimen ID printer label (with or without barcode) to sterile screw-capped tube. Write “super” or “supernatant” on the label to distinguish it from the tube containing the spun sediment.

3. Aseptically and gently decant the upper portion of the centrifuged fluid into the “supernatant” tube. The portion remaining, ~0.5 ml, is the sediment and is used for preparing the smear and for inoculating the media.

4. Agitate the sediment to re-suspend any cellular material and microorganisms.5. Using sterile inoculating loop or sterile pipette, remove a portion/drop and place

onto a glass slide. Do not spread the fluid. Occasionally a second drop of fluid can be added to the same area to increase the concentration of the fluid for examination.

Dried material or very small amounts of clinical specimen

1. Emulsify the specimen in 0.5ml of sterile broth or saline, vortex if necessary.2. Use a sterile 10µl loop or Pasteur pipette to transfer 1 drop to a slide and spread

into an even thin film.

Biopsy and tissue sections

A touch preparation is preferred: place the tissue into a sterile petri dish and slice or mince using sterile scalpel. Using sterile forceps, gently touch the glass slide with one or more of the sample pieces.

For times when there is no other option, grind the sample using a sterile tissue grinder. Spread one drop on the slide and spread to the size of a dime. Grinding the sample before the smear is made will often destroy characteristic cellular structure and bacterial arrangements.

Record the method of preparation (e.g. “touch” for touch preparation) on the slide and on the requisition.

Broth Cultures

Using a Pasteur pipette, transfer 1 or 2 drops to a glass slide and spread into an even film.

Colonies from Solid Agar

1. Place a drop of sterile saline on a slide.2. Transfer a small portion of the colony with an applicator stick or inoculating loop.3. Gently mix to emulsify.

Urine Specimens

Place 10µl of well mixed, uncentrifuged urine onto a glass slide.

Smear Fixation

1. Allow the smears to air dry2. After the slides are air dried, slides should be fixed on the slide warmer (the slide

should not be hot, just warm to the touch) for a few minutes3. Allow slides to cool before staining

Staining and Interpretation of Smears [Preparing a smear] [Gram stain procedure and examination] [Negative staining] [Spore staining [Observation of living bacteria]

Important information such as shape and degree of motility can be obtained by observation of living bacteria with the phase contrast or dark field microscope. However bacteria are routinely stained with different dyes in order to reveal different properties and to enhance contrast for viewing with conventional bright field microscopy. A number of stains have been developed to distinguish spores, nuclear bodies, capsules, and characteristics of the cell wall.

The staining methods we will use kill the bacteria, reducing the risk of infection by pathogenic organisms. Since the rigid cell walls of bacteria prevent distortion of morphology upon drying, samples can be spread onto a glass slide and air dried, then fixed to the surface by passing the slide quickly through a flame, melting the complex carbohydrates of the cell walls to the glass and killing the cells.

The Gram stain is routinely used as an initial procedure in the identification of an unknown bacterial species. Bacteria bear a slight net negative charge and usually bind positively charged dyes such as methylene blue and crystal violet. A species can be classified as Gram positive, Gram negative, or Gram variable depending on the ability if cells to retain the blue dye. Gram negative bacteria do not retain the dark blue color, but can be counterstained a light red so that they can be seen in bright field microscopy. Since two dyes are used to distinguish types of bacteria, Gram staining is called a differential staining method.

The Gram stain is a direct method, since the cells themselves retain dye. In indirect, or negative, staining, smears are produced by mixing material with India ink or acidic dyes such as nigrosine. Acidic dyes have a negative charge and are repelled by the negatively charged cell walls. Cells remain unstained against a dark background.

Some species produce spores, which are dormant cells with thickened cell walls. Spores are often detectable in Gram stains or by phase contrast microscopy of living cells, however differential staining methods may be necessary to confirm or reject the presence of spores in a culture. As with the Gram stain, a spore stain distinguishes spores on the basis of cell wall properties.

In the laboratory you will practice the Gram stain technique on a variety of Gram positive and Gram negative bacteria grown on different types of media. You will learn the technique of negative staining with nigrosine dye in order to clearly distinguish shapes of bacteria. You will also carry out spore staining on appropriate cultures. Finally, you will observe living bacteria to become familiar with features that can be seen without staining, including cell shapes, patterns of association, and motility.

Preparing a smear

A properly prepared smear accomplishes two things. It causes bacteria to adhere to a slide so that they can be stained and observed. It also kills them, rendering pathogenic bacteria safe to handle. An objective in preparing smears is to learn to recognize the correct density of bacteria to place on the slide. Too many, and they overlap each other giving false positives or crowding each other to make a mess. Too few, and they cannot be located on the slide.

A circle should be marked on the under side of a slide with a glass etching tool. Several circles can be located on the same slide.

The slide must be grease-free. A good way to clean a slide is to repeatedly breathe on it, followed by rubbing vigorously with a Kimwipe or paper towel to remove the fog. When the slide de-fogs immediately after breathing on it, it is sufficiently clean.

To prepare a smear from a dry culture, a very small drop of distilled water should be placed over the circled area. After aseptically removing material from a culture it is them mixed with the drop or placed directly on the slide if it is a dilute broth culture. It takes very little material to produce a successful smear.

The drop is air-dried completely, which takes a short time if a small drop is prepared.

While holding the slide with a clothes pin it is quickly passed it through a flame. Three quick passes are usually sufficient to kill the bacteria and cause them to adhere.

After cooling the slide, the staining procedure is conducted.

How to screw up a smear Forget to clean the slide Use too much material - suspension should be just barely cloudy Use so much liquid that it takes forever to dry Heat the smear before letting it air dry, boiling the bacteria instead of attaching

them Overheat the smear, melting cell walls and possibly breaking the slide

The Gram stain

In Gram staining bacteria fixed to a slide are treated with a basic dye that binds electrostatically to the negatively charged cells. Next, the preparation is treated with a

mordant such as iodine to form an insoluble dye-iodine complex. The slide is then washed with alcohol to solubilize and remove the dye-mordant from Gram negative cells but not Gram positive ones. Differential extraction of the dye-mordant by the decolorizing agent is the critical step that distinguishes the bacteria. A counterstain, safranin, is applied in the final step. Cells that have been decolorized will take up the second basic dye whereas those already stained with the first dye will not.

The mechanism of the differential staining response has not been resolved with certainty. One theory holds that differences in the cell wall chemical composition account for the staining response. A second theory maintains that the thicker walls of Gram positive bacteria are dehydrated by the decolorizing solution and shrink, resulting in the closure of pores in the wall, trapping the dye-mordant within the cell. The thinner cell wall of Gram negative bacteria would be readily penetrated by the decolorizer. What is known with certainty is the critical role of the cell wall. Removal or alterations of the wall from Gram positive organisms converts them to Gram negative cells.

Since many Gram positive bacteria tend to become Gram negative with age, the Gram stain should be used with overnight cultures. Sample from the edge of a colony, where cells are actively growing.

The stains you use in this laboratory will stain hands and clothing as well as bacteria. Do not wear good clothes in the laboratory, wear gloves when staining, and keep in mind that all stains are assumed to be toxic unless otherwise stated.

Components

Different formulas have been used for crystal violet, safranin, and decolorizer, all of which are effective.

1. Gram's crystal violet: 1% aqueous crystal violet dye; [Hucker's crystal violet] 2 g crystal violet 90% dye content, 20 ml ethyl alcohol, 0.8 g ammonium oxalate, 80 ml distilled water

2. Gram's iodine: 1 g iodine, 2 g potassium iodide, 300 ml distilled water3. Gram's safranin: 4 g safranin powder, 200 ml ahydrous ethanol, 800 ml distilled

water4. Gram's decolorizer: 25% acetone, 75% isopropyl alcohol

A heat-fixed bacterial smear is first covered completely with a few drops of a solution of crystal violet, a purple basic dye.

After 30-60 sec the smear is rinsed with water by squirting the slide above the smear and letting the water wash over it until the water runs clear.

Several drops of iodine (the mordant) are applied to cover the smear and left for 60 sec., then rinsed again.

A few drops of an isopropanol-acetone mixture or similar solvent are added at a time until the wash is colorless, then the slide is rinsed again.

A red basic dye, aqueous safranin, is applied for 30-60 sec.followed by a rinse.

The smear is blotted (not wiped) to remove excess water, using bibulous (absorbent) paper or a paper towel. The slide is then air dried the rest of the way.

How to screw up a Gram stain Screw up the smear to start with Apply stain to the wrong side of the slide Stain or rinse the smear incompletely Allow stain to dry up before rinsing Forget to decolorize or decolorize incompletely Rub off the material when you blot it

Microscopic observation of a Gram stain

A good approach to observing any smear of very minute objects is to examine it under low power (40x) in bright field to become oriented. After focusing, one then works up to 100x, 400x, and finally oil immersion (100x). The immersion oil is placed directly on the smear. By the way, the focal lengths of the high dry and oil immersion lenses are less than the thickness of a slide. The smear must be up, or focusing won't be possible. The top of the slide can be identified by feeling for the etched circle on the bottom of the slide.

At low magnifications Gram stained material looks like dirt on the slide. Higher magnifications are needed in order to see any detail at all. Bacteria are often concentrated in a ring around the original smear. Bright field oil immersion microscopy is necessary to see an undistorted image of any directly stained bacterial smear. A Gram negative or positive phenotype cannot be confirmed with certainty using only a dry magnification, since typical cells are a half micrometer in diameter, less than the best resolution of the high dry lens. Phase contrast or dark field viewing improve the resolution, but both distort the color. High dry magnification distorts both shape and color.

After putting immersion oil on a slide, the high dry (40x) lens can't be used again unless the oil is removed. Slides are usually blotted to remove excess oil, then dipped in xylene several times to dissolve the oil film, and air dried in the fume hood.

Indirect (negative) staining

The acidic dye nigrosine will be used to visualize the capsule or sheath that surrounds some bacteria in a process called negative staining. Capsules are composed primarily of polysaccharides or glycoproteins and are gelatinous in texture. They are readily destroyed by heating and hence direct staining methods cannot be utilized. In general, the size and shape of microorganisms is often less distorted with indirect staining procedures, especially when sampled from a broth culture. Therefore negative staining is useful whenever the morphology of individual bacteria is in question. Morphology can often be determined with confidence with only the high dry lens. Consider that this procedure does not necessarily kill the organism, so be careful.

After preparing a clean, greaseless slide, a small drop of nigrosine is mixed with a small drop from a broth culture or with a quantity of dry material.

The drop is spread across the slide using the edge of another slide as a spreader. This same procedure is used for blood smears.

After air drying, the smear is observed using the high dry lens, or oil immersion if desired. The smear will be most dense where the nigrosine dye was deposited on the slide. Somewhere along the tapered spear the density will be ideal. The background should be blue-gray. Bacteria will be evident by the absence of any color.

Spore staining

The following modification of the Wirtz method, described below, has been effective and trouble free in our experience. You are cautioned, though, that the color may fade after a few days. The result is a delicate combination of green and red that is readily recognized provided lighting is optimum. ***CAUTION*** Malachite green dye is toxic, and breathing of fumes or contact with skin can be hazardous. This procedure should be carried out in a fume hood. The stand, burner, dyes and pipets will be available in the hoods. The procedure is messy - wear a lab coat or old clothes.

A smear is prepared and fixed with 20 passes through a flame. The smear is placed on a screen well above a moderate flame from a bunsen

burner. A generous amount of saturated aqueous malachite green is applied to the slide

and allowed to steam off for 10 to 20 minutes. Ideally, the heat is maintained so that the dye barely steams, that is, whitish vapors are barely visible. Dye must be added so that the liquid does not dry up.

After cooling the slide is rinsed with tap water to remove excess stain. The slide is then counterstained for a minute in 0.25% aqueous safranin. The slide

is then rinsed, blotted, and dried.

Spores stain a light green,while the rest of the cell stains pink. Spores are best seen with oil immersion microscopy. Often, the colors are not very strong, so it is necessary to have the microscope in good alignment with optimum contrast and lighting. Make color notes right away, as the green may fade after a few days.

Observations on living bacteria

Sometimes assay results are compromised because a contaminating organism grows in the medium instead of the intended bacterial isolate. For a quick check to verify that cell morphology is consistent with the culture from which the inoculum was taken, a wet mount can be prepared and examined in dark field and/or phase contrast. If present, endospores are often evident in phase contrast, allowing one to avoid having to do a spore stain.

Very often, identification of an unknown organism requires knowledge of its motility, that is, its capability for translational movement. The results of motility agar incubations can be difficult to interpret, partically for aerobic bacteria that don't grow well deep into the agar. A good quick check for motility is to examine a very young culture using the hanging drop method. A young culture would be a broth culture inoculated the night before, or a broth culture that was diluted 10 fold or so in the morning, incubated, and examined in the afternoon. A hanging drop culture is prepared by placing a very small drop of medium on a coverslip, then inverting the coverslip over a depression slide so that the bottom of the drop does not make contact with the slide itself. Vaseline can be used if necessary, to make a sealed chamber.

Hanging drops can be examined using all objective lenses, although to be able to look throughout the depth of the drop the limit may be 100x. The curved depression slide will distort the effects of phase contrast, but dark field may work and will be sufficient to detect movement. All live bacteria move by Brownian (molecular) motion, at a vibration rate that is inversely proportional to the size of the cell. Rapid Brownian movement is a common characteristic of non-motile cocci such as Staphylococcus, Streptococcus, or Micrococcus. However some bacteria are flagellated, and exhibit translational movement as well. Truly motile organisms will zip across the microscope field. Look for definite directional motion, tumbling, and movement against currents.

Dispose of wet mounts carefully, since the bacteria will be viable.

Gram stainingFrom Wikipedia, the free encyclopedia

Gram-positive anthrax bacteria (purple rods) in cerebrospinal fluid sample. If present, a Gram-negative bacterial species would appear pink. (The other cells are white blood cells).

A Gram stain of mixed Staphylococcus aureus (Gram positive cocci) and Escherichia coli (Gram negative bacilli), the most common Gram stain reference bacteria

History

The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who

developed the technique while working with Carl Friedländer in the morgue of the city hospital in Berlin. Gram devised his technique not for the purpose of distinguishing one group of bacteria from another but to enable bacteria to be seen more readily in stained sections of lung tissue.[2] He published his method in 1884, and included in his short report the observation that the Typhus bacillus did not retain the stain.[3]

Uses

Gram staining is a bacteriological laboratory technique[4] used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative) based on the physical properties of their cell walls.[5] Gram staining is not used to classify archaea, formally archaeabacteria, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.[6]

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, however. It is of extremely limited use in environmental microbiology, and has been largely superseded by molecular techniques even in the medical microbiology lab. Some organisms are Gram-variable (that means, they may stain either negative or positive); some organisms are not susceptible to either stain used by the Gram technique. In a modern environmental or molecular microbiology lab, most identification is done using genetic sequences and other molecular techniques, which are far more specific and information-rich than differential staining.

Medical

Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much more quickly than culture, and is especially important when infection would make an important difference in the patient's treatment and prognosis; examples are cerebrospinal fluid for meningitis and synovial fluid for septic arthritis.[4][7] .

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which are stained purple by crystal violet, whereas Gram-negative bacteria have a thinner layer (10% of cell wall), which are stained pink by the counter-stain. There are four basic steps of the Gram stain:

applying a primary stain (crystal violet) to a heat-fixed (death by heat) smear of a bacterial culture

the addition of a trapping agent (Gram's iodine) rapid decolorization with alcohol or acetone, and counterstaining with safranin.[8] Basic fuchsin is sometimes substituted for

safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain.[9]

Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.

Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and, therefore, color the cell.[10]

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative cell will lose its outer lipopolysaccharide membrane, and the inner peptidoglycan layer is left exposed. The CV–I complexes are washed from the Gram-negative cell along with the outer membrane. In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CV–I complexes become trapped within the Gram-positive cell due to the multilayered nature of its peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left on too long (a matter of seconds).

After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color. [11]

[12]

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of Bacillus, Butyrivibrio, and Clostridium, a decrease in peptidoglycan thickness during growth coincides with an increase in the number of cells that stain Gram-negative.[13] In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

Examples

Gram-negative bacteria

The proteobacteria are a major group of Gram-negative bacteria. Other notable groups of Gram-negative bacteria include the cyanobacteria, spirochaetes, green sulfur, and green non-sulfur bacteria.

These also include many medically relevant Gram-negative cocci, bacilli, and many bacteria associated with nosocomial infections.

Gram-positive bacteria

In the original bacterial phyla, the Gram-positive forms made up the phylum Firmicutes, a name now used for the largest group. It includes many well-known genera such as Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium. It has also been expanded to include the Mollicutes, bacteria like Mycoplasma that lack cell walls and so cannot be stained by Gram, but are derived from such forms.

Gram-indeterminate bacteria

Gram-indeterminate bacteria do not respond to Gram staining and, therefore, cannot be determined as either Gram-positive or Gram-negative

Isolation & Gram Stain Preparation

Introduction:

     Since microorganisms live as mixed cultures in nature, we need to be able to isolate them into a pure culture for study.  The most common method to do this is the streak plate method.  In this lab you will streak 2 colonies from each of the sample plates for isolation.  The streak plate method dilutes the sample mechanically by dragging the loop across the agar surface, separating the organisms.  The loop is flamed between each region to help dilute each region further.

     Bacteria can also be isolated by using specialized media.  These help the microbiologist inhibit growth of unwanted organisms or visually identify likely organisms.  Selective media contain stubstances to inhibit the growth of 1 group while permitting the growth of others.  Columbia CNA agar contains the antibiotics colistin and nalidixic acid to inhibit the growth of Gram negative organisms.  It is selective for Gram positive organisms.

     Differential media causes an observable color change, differentiating the bacteria that are able to carry out that specific biochemical reaction.  Enrichment media enhances the growth of certain organisms.  Some media are both selective and differential.  EMB (Eosin Methylene Blue) agar is selective for Gram negative organisms and is differential for certain Gram negative enteric bacteria.

     Escherichia coli:       large blue/black colon, green metallic sheen

     Enterobacter & Klebsiella:     large, mucoid, pink/purple colonies, no metallic sheen

     Salmonella, Shigella, Proteus:  large colorless colonies

     Shigella:  colorless to pink colonies

Isolation using different media:

Select 2 different colonies from each of the finger culture, hair culture, and air culture and streak for isolation.  You will prepare a streak for isolation a plate of Columbia CNA agar and EMB agar for each sample colony. For each colony you will streak ½ of a CNA plate and ½ of a EMB plate.

Label your plates and incubate at 37degrees Celsius.

Gram stain preparation:

     Bacteria are not easily visualized under a microscope without staining.  Stains allow a greater contract between the organism and its background, allow us to identify the morphology (shape, arrangement, Gram reaction), and see flagella, capsules, endospores, etc.  Dyes are stains are generally divided into basic and acidic dyes. 

     The Gram stain is the most common stain used in microbiology, and is used to differentiate between Gram positive and Gram negative bacteria.  Gram positive bacteria stain purple, and Gram negative stain pink.  The difference in Gram reaction is due to differences in cell wall structure.  The Gram positive cell wall is 60-90% peptidoglycan.  The Gram negative cell wall only has 2-3 layers of peptidoglycan surrounded by an outer membrane of phospholipids, lipopolysaccharide, lipoprotein, and proteins.  Only 10-20% of the Gram negative cell wall is peptidoglycan.

Heat fixation from agar medium:

1.  Place ½ a drop of distilled water on a clean slide.

2.  Use aseptic technique to transfer a small amount of culture from the agar surface and touch it to the water until it turns cloudy.

3.  Burn the remaining bacteria off the loop.

4.  Use the loop to spread the suspension over a large portion of the slide for form a thin film, and allow it to completely air dry.

5.  Pass the slide film side up through the flame of the burner 3-4 times to heat fix.  Be careful not to use too much heat that may distort the organisms.  Too much heat may cause a Gram positive organism to stain Gram negative.

Heat fixation from a broth culture:

1.  Aseptically place 2-3 loops of the culture on a clean slide.

2.  Spread the suspension over the slide and allow to air dry.

3.  Use the burner to heat fix as described above.

Gram stain procedure:

1.  Stain the bacteria with the basic dye crystal violet.’(1 minute)

2.  Cover the smear with Gram’s iodine solution.  This allows better retention of the stain by forming an insoluble crystal violet iodine complex. (1 minute)

3.  Decolorize the smear with the Gram’s decolorizer, a mixture of ethyl alcohol and acetone.  Gram positive bacteria will retain the crystal violet complex while Gram negative are decolorized. (just until purple stops flowing, and wash with water immediately)

4.  Counterstain with the basic dye safranin.  Gram positive bacteria are not affected by the counterstain because they are already purple.  Gram negative bacteria are colorless, and will become directly stained by the safranin.  (2 minutes and then wash with water).

Evaluate your skill in streaking for isolation with the sample colonies:

Results:  Note how many different colonies it appears  from each sample colony you streaked.

http://inst.bact.wisc.edu/inst/images/book_3/chapter_14/14-2.jpg

Example Gram Stain Results:Gram positive rods:

http://lib.jiangnan.edu.cn/ASM/115-8.jpg

Gram positive cocci:

http://media-2.web.britannica.com/eb-media/05/58705-004-12AFD703.jpgGram Negative Rods:

http://jon9783.myweb.uga.edu/image/image1.jpgGram Negative Cocci:

http://lib.jiangnan.edu.cn/ASM/118-1.jpg

http://wendysmicrobiology.blogspot.com/2009/08/isolation-gram-stain-preparation.html