Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - Arizona University
Demonstrating Author: Luisa Ikner
The spectrum of research in environmental microbiology is broad in scope and application potential. Whether the work is bench-scale with known bacterial isolates, or in the field collecting soil or water samples containing unknown bacterial isolates, the ability to quickly and visually discern culturable populations of interest remains of great import to environmental microbiologists even today with the abundance of molecular techniques available for use. This video will demonstrate one such technique, known as Gram staining.
The Gram stain is a classical and important staining technique that remains widely used by environmental microbiologists. Similar to a simple stain, it allows for assessment of bacterial cell morphology (e.g., cocci, rods, spore-formers), size, and arrangement (e.g., chains or clusters). In addition, it allows for differentiation of bacteria into two principle distinct groups — Gram-negative and Gram-positive — according to cell wall composition and structure (Figure 1).
Gram staining is a multi-step process. Prior to staining, a bacterial smear is prepared using a plate, slant, or broth culture. The smear prep is dried and fixed onto a clean glass slide. A primary stain of crystal violet is then applied to the fixed smear. Crystal violet is a basic stain comprised of positively charged colored ions (i.e. chromophores) that form weak ionic bonds with negatively charged functional groups present in the bacterial cell wall. After gently rinsing the slide with water, Gram’s iodine is applied, and forms insoluble complexes with the crystal violet in the cell wall. The crystal violet-iodine complexes further bind with peptidoglycan, a principle component of bacterial cell walls. Following a second water rinse, a decolorizing agent is briefly applied to the smear. For Gram-negative bacteria, the crystal violet-iodine complex is washed away during the decolorizing step, with Gram-positive bacteria retaining the purple stain. A third and final water rinse is followed by a counterstain of safranin that colorizes Gram-negative bacteria pink or red.
Figure 1. Comparison of the cell wall of Gram-positive and Gram-negative bacteria.
1. Sample Collection
- Collect soil sample and transport to the laboratory for microbial analysis.
- In the lab, weigh a 10 g sample using an analytical balance.
- Dilute the sample 1:10 into 95 mL of phosphate-buffered saline (10 parts soil is equivalent to 5 parts aqueous liquid), and vortex to mix (Figure 2, Step 1).
- Perform subsequent 1:10 dilutions up to at least 10-5 g soil per mL, and spread-plate selected dilutions in replicates of two or three onto a low nutrient agar medium (e.g., R2A) (Figure 2, Steps 2-3).
- Incubate the plates for one week at room temperature (Figure 2, Step 4).
- Select one or two colonies for isolation, and streak onto fresh agar plates (Figure 3, Steps 1-3).
- Incubate the streak plates for two to three days at room temperature (Figure 3, Step 4).
2. Preparation of Bacterial Smears
- Observe the streak plates for isolated colonies.
- To prepare each smear prep, dip an inoculating loop into ethanol, flame-sterilize, and place 1 to 2 loopfuls of sterile distilled water onto the center of pre-cleaned glass slides.
- Sterilize the inoculating loop again as previously described. Once cooled, remove a small amount of culture from a single isolated colony and mix it with the water droplets on the slide (the smear should resemble diluted skim milk). The inoculating loop must be cooled prior to colony isolation. A loop that is too hot will cause the colony and/or medium to splatter, which may lead to aerosolization of bacteria. Generally, when the loop is too hot for use, a “hissing” sound will be heard when applied to the agar or colony. Improper cooling of the loop may also result in less efficient culture-to-slide transfer, and distortion of cell morphology.
- Spread the smear over the surface of the slide measuring approximately 2.5 cm x 2.5 cm, and allow it to air dry. It is important for air drying to occur under laminar flow conditions. Slides should not be blown dry so as not to disrupt the smear. Also, slides must not be flame-dried, in order to maintain cell morphology.
- After drying, heat fix the smear by passing the slide quickly through a flame 2-3x. The slide should not be held stationary in the flame, to prevent distortion of cell morphology and/or damage to the glass slide.
3. Gram Staining
- Secure the slide at one end using a clean clothespin.
- Cover the smear with crystal violet (primary stain) and hold for 2 to 3 min.
- Carefully wash the slide with distilled water. The water stream should not be directed at the smear in order to prevent damage and/or detachment from the glass slide.
- Cover the smear with Gram’s iodine and hold for 2 min, then gently rinse the slide with water.
- Decolorize the smear using 95% ethanol until stain no longer washes from the slide (this usually takes no more than 20 s depending on the thickness of the smear), then immediately rinse with distilled water. This step is critical to avoid over decolorizing the slide, which may lead to a false Gram stain designation (i.e., Gram-variable).
- Add the counterstain (safranin) to the smear and hold for 30 s. Then gently rinse the slide with distilled water and blot dry using absorbent paper.
4. Microscopic Observation of Slides
- Observe the slides using low (e.g., 4X or 10X), high-dry (e.g., 40X), and oil immersion (100X) objectives. For oil immersion, add the oil directly to the smear.
- For representative results of Gram-positive and Gram-negative soil bacteria, see Figures 4 and 5.
Figure 2. Dilution and Spread-Plating Technique. Please click here to view a larger version of this figure.
Figure 3. Colony Isolation Using the Streak Plate Technique.
Figure 4. Gram-positive soil bacterium Staphylococcus aureus.
Figure 5. Gram-negative soil bacterium Escherichia coli.
Gram staining allows for quick visualization of bacterial morphology and broad cellular distinction from a wide range of environmental samples. To stain bacteria, a uniform smear is applied to a glass side and allowed to dry. After heat-fixing the smear, crystal violet is applied.
A decolorizing agent rinses away the crystal violet from Gram-negative cells, but not Gram-positive cells. A second dye, typically safranin, is used as a background stain to visualize the Gram-negative cells. Once stained, the cells can be assessed for cell morphology, size, and arrangement, such as chains or clusters.
This video will demonstrate how to prepare an environmental sample, isolate the bacterial species found therein, and perform a Gram stain on the isolated colonies
Gram staining allows for the categorization of most bacteria into two broad structural classes: Gram-positive and Gram-negative. While both classes have an underlying phospholipid plasma membrane, the structure of the cellular wall varies greatly. The Gram-positive cell wall is primarily composed of a thick layer of peptidoglycan, which is a polymer that consists of sugars and amino acids. Gram-negative cell walls have a thinner layer of peptidoglycan, sandwiched between a second lipid membrane. This outer membrane typically contains lipopolysaccharides.
The positively-charged crystal violet binds weakly to the negatively-charged bacterial cell wall. Gram’s iodine forms an insoluble complex with the crystal violet dye, thereby fixing it in the cell wall.
During the decolorizing step, the peptidoglycan in the Gram-positive cells is dehydrated, causing it to contract and trap the crystal violet-iodine complexes. In Gram-negative cells, the decolorizing agent compromises the outer membrane, increasing its porosity. This allows the crystal violet-iodine complexes to be washed away.
Now that you understand the principles behind Gram staining soil bacteria, let's see the process performed on soil bacteria in the laboratory.
After collecting a soil sample in the field, bring it into the laboratory for analysis. Refine the sample with a sieve, and weigh 10 g of the sieved soil using an analytical balance.
Dilute the sample into 95 mL of phosphate-buffered saline and vortex to mix. Perform additional 1 to 10 dilutions, vortexing between each dilution. Transfer aliquots of at least 3 successive dilutions, onto replicate low nutrient agarose plates.
Following ethanol-flame sterilization and cooling of a bent glass rod by tapping onto the media, spread the sample over the surface of the plates. Incubate the plates at room temperature. After incubating for 3 to 5 days, select the highest dilution with 30 to 300 discrete colonies. With a sterile inoculating loop, select colonies of interest for isolation.
Streak the colony onto one section of a fresh plate. Sterilizing the loop between streaks, make successive streaks in a zig-zag pattern onto each section of the plate to allow for subsequent isolation of discrete single colonies. Incubate the plates for 1 to 2 days at room temperature.
To begin preparation of bacterial smears, attach a clip to a pre-cleaned glass slide for ease of handling. For each bacterial smear, clean and flame-sterilize an inoculating loop. Place 2 loopfuls of sterile distilled water onto the center of the slide.
After sterilizing the loop again, remove a small amount of culture from an isolated colony, and mix with the water on the slide. It's important to cool the loop before touching the culture by tapping an uninoculated portion of agar several times. The smear should resemble diluted milk. Allow the slide to dry at room temperature. After drying, heat fix the smear by quickly passing it through a flame.
Once dry, pipette crystal violet onto the smear, and let sit for 2 - 3 min. Carefully rinse the slide with distilled water by aiming the direct flow towards the top of the slide, allowing the water to gently flow down. Do not aim the water flow directly at the smear.
Cover the slide with Gram's iodine. After 2 min, gently rinse with distilled water. Decolorize the slide with 95% ethanol until stain no longer washes away. Immediately rinse with distilled water. This will limit over-decolorizing the smear.
Add safranin as a counter-stain to the smear for 30 s. This will stain any Gram-negative cells present. Gently rinse with distilled water and blot dry with absorbent paper. Observe the resulting slide with a microscope. Use a low power objective first to make coarse adjustments and find an ideal portion of the smear before moving on to the smaller field-of-views of the higher magnifications.
After further imaging and adjusting the smear with a medium power objective, add immersion oil directly to the smear. The oil is needed for high power objectives that will provide the best micrographs. Gram-positive bacteria will appear blue or purple in color, while Gram-negative cells will be red or pink. In addition to cell wall structure, shape and arrangement are elucidated from the resulting micrographs.
The ability of Gram staining to qualitatively study bacteria is important to a wide range of scientific fields.
Soil is just one of the environmental sources from which bacteria can be isolated and analyzed. For drinking water, sample preparation must be modified. Samples of tap water can be drawn from a faucet, and plated onto growth media that facilitates the growth of diverse, heterotrophic bacterial colonies. Upon plating onto a medium such as R2A, the process is nearly identical to the soil procedure.
For certain bacterial identification techniques, the parameters are dependent on the cell wall type of the bacteria of interest. In this example, a septic patient's blood was tested and was found to be harboring Gram-positive bacteria.
With this information, species-specific peptide nucleic acid probes were chosen that would bind to the cells' rRNA. These probes were bound to fluorescent dyes that were used to identify the species present.
Because of the fundamental differences in Gram-positive and -negative cellular structure, bacteria have unique responses to other compounds besides crystal violet. This experiment sought to isolate Clostridium difficile, a Gram-positive bacterium, from fecal samples. Cycloserine, which inhibits the growth of Gram-negative cells, was added to the agar plate. The Gram-positive cells that grew on the plate were further isolated via other methods.
You've just watched JoVE's introduction to Gram staining for environmental studies. You should now understand the benefits of the process and how to perform the technique and utilize the results. Thanks for watching!
Applications and Summary
The Gram stain is used in the many sub-fields of both environmental and clinical microbiology. Water quality scientists may use the Gram stain as a confirmatory tool for the detection of fecal bacteria in water samples. Bacterial isolates from soils are Gram stained in order to further characterize culturable soil communities. For environmental microbiologists, Gram stain aids in the categorization of bacterial populations according to cell wall structure. This, in turn, provides information about the general ability of a given microbial community to withstand desiccation and other environmental stressors. Knowledge of Gram stain designation is also of importance in the research and development of disinfectants and other antimicrobials, as Gram-positive bacteria tend to be more resistant to inactivation by particular chemistries than Gram-negative bacteria.
For clinical microbiology applications, the Gram stain is used to confirm the identity of bacteriological disease agents along with traditional diagnostic methods. It is also of great assistance when culturing has failed, or is not an option. Gram staining of clinical specimens can reveal the presence of etiologic agents that may not have been observed otherwise.