Performing a Gram Stain

The Gram stain has been in existence for more than 100 years, and remains a key starting point to identify microbial species. The stain makes use of the differing membrane structures between Gram positive (single cell membrane with a tough outer cell wall of peptidoglycan), and Gram negative organisms (have two layers of membranes, with a thin layer of peptidoglycan sandwiched between them).

Materials | Preparing the Slide | Staining the Slide | Microscopy | Interpretation

Materials:

Glass slides
Grease pencil (optional, but helps identify where you placed organisms).
Sterile saline or water -- if you don't have either available, try a local drug store for saline, or make certain to Gram stain your liquid source to see if it is contaminated, else you might mistake organisms in non-sterile water for those you are trying to identify.
Inoculating loop or sterile toothpicks.
Heat-source to fix bacteria to the slide -- can be a bunsen burner, or a hot plate set to 42°C (108°F).

If using a hot plate, it's important that it not get hotter than 42°C else you may lyse (rupture) the bacteria from the heat.

Forceps - to hold the slide while heat fixing, and during staining.
Source of microbes - can be from a both or plate culture, or from a direct environmental source - such as swab of the inside of your mouth or the local water fountain.
For the staining - the following reagents can be purchased from many biological supply houses, and work best of they're placed in small squirt bottles (100-200mL). Handle carefully as they will *irreversibly* stain clothing.

Crystal violet: Per 100mL:

Add 2g crystal violet powder to 20mL ethanol.
Next add 0.8g of ammonium oxalate to 80mL water.
Mix the two solutions.

Lugol's Iodine:

Add 2.0g of potassium iodide (KI) to 100mL of water.
Next add 1.0g of iodine crystals (note elemental iodine is toxic and should be handled within a fume hood).
Store in a brown or light-blocking container.

Decolorizing agent: can by acetone or 95% ethanol (denature EtOH works).

Saffranin (red counterstain):

0.25g of Saffranin O powder added to 10mL ethanol.
Add to 90mL water and mix for use.

Squirt bottles: Four small squir bottles ~100-200mL in volume are useful for holding each of the staining components.
Water - tapwater can be used to wash slides during staining.
Tray (glass, plastic or metal) with rack, on which you will place the slides, and collect used dyes and wash.
Paper towels or blotting paper (sometimes called "bibulous" paper).
Microscope - needs to have a 100X oil objective to appropriately view and interpret the slides.
Immersion oil for viewing bacteria at 100X magnification.

Preparing the slide:

Prepare a slide with your microbial source: Make a small circle with the grease pencil (~1.0cm in diameter), if you have one available. If not, note where on the slide you place your inoculum. Magic markers shouldn't be used as the decolorizing agents (ethanol or acetone) dissolve the inks.
Microbial source - remember, less is more! One common mistake made by budding microbiologists it to put multiple colonies of bacterial on the slide to stain.. this frequently causes everything to stain purple (Gram positive) as subsequent dyes and decolorizing agents can not penetrate the microbial mass.

If collecting material from an environmental source, whet a swab with sterile saline or water and swab your area. A good introductory exercise is to Gram stain for bacteria in your mouth. In this case, swab the inside of your cheek and areas between your teeth and gum-line.. lots of microbes! Swab within the circular area you just drew on the slide.
From broth cultures, sterilize an inoculating loop, place in your culture to that liquid is held by capillary action within the loop, and transfer this amount to the slide -- all you need is a few microliters. Most inoculating loops will hold 1 or 10uL, which is more than adequate.
If from a culture plate, place a small drop of saline or water within the circle on the side (<10uL). Touch a colony on the place with a sterile inoculating loop or toothpick. Transfer the collected material to the drop of liquid on the slide - again, less is more -- if you can see clumps of material from the colony, you probably have too much.

Heat-fix this slide. This step makes certain the bacteria stick to the glass during staining. If you don't perform it the bacteria may be washed off during staining.

Bunsen Burner Method: Light the bunsen burner. Hold the slide with a pair of forceps (strong tweezers) so you don't burn your fingers. Pass it through the interface bewteen the blue flame and the yellow flame - this is the hottest region of the flame - 5 times. The slide should sit in this region for no more than a second, as if it gets too hot the bacterial will rupture, and you will not get a good stain.
Heating Plate: If using a heating plate, set it to 42°C (~108°F). Place your glass slide on the hotplate until all the liquid evaporates.

Staining the slide:

Gram Positive	Gram Negative	Step in the Staining
		0. Start with a heat-fixed slide of your material. Recall, Gram positive bacteria have a single cell membrane and hardy outer cell wall made of peptidoglycan. In contrast. Gram negative cells have two membranes. In between the two membranes is the periplasmic space which contains a layer of peptidoglycan. <-- Follow the images to the right to see how Gram positive and Gram negative cells would appear during each stage of the stain.
		1. Crystal Violet: Flood the slide with Crystal violet. Let sit for 30 seconds, then rinse with tap water. If available, use forceps to hold the slide while rinsing. The crystal violet binds to the membrane of Gram positives, and the outer-membrane of Gram negative bacteria.
		2. Lugol's Iodine: Now flood the slide with the Lugol's iodine, which forms a strong complex with bound crystal violet. Let sit for 30s, then rinse the slide with tap water. A this stage both Gram positive and Gram negative bacteria will stain purple.
		3. Decolorization -- this is the most important step. Ethanol tends to be more forgiving than acetone, and may be worthwhile to try for beginners. Acetone or 95% ethanol dissolves the outer membrane of Gram negative bacteria, but not Gram positive bacteria. If done properly, Gram positive bacteria remain purple at this stage, while Gram negative bacteria become colorless. However, too much decolorization and all bacteria will appear Gram negative (all crystal violet+iodine will be washed away); too little, and all bacteria will appear Gram positive (not enough to remove the outer membrane of Gram negatives). In general, hold the slide at a slant and count for 3 seconds while squirting top edge of the slide with the decolorizing agent, so it runs down the length of the slide. Then immediately rinse in tap water to remove remaining decolorzing agent.
		4. Counterstain with Saffranin: Flood the slide with the dye and let sit for 30s, then wash. Saffranin is a red dye and actually stains both Gram positive and negative bacteria. However, for Gram positive bacteria that are already purple, they remain an intense purple color while previously colorless Gram negative bacteria take up the red stain and will appear red by microscopy.
Purple	Red	5. Interpretation (more below): Slides need to be read at 100X magnification on a microscope. Lenses at this magnification require immersion oil to obtain clear imaging of material on the slide. Gram positive organisms will be dark purple, while Gram negative will appear bright red.

Microscopy:

Blot the stained slide on blotting paper - make certain to blot, not rub the slide dry. Rubbing will rub the bacteria off the slide. If you don't have blotting paper, lens paper or study paper towel that doesn't shed fibers can be used.

Next place the slide on the microscope stage and first observe at low magnification (4X or 10X). If you used a grease pencil, you can focus on the grease line to focus on the plane where the stained bacteria will be. If you didn't use a grease pencil, most slides have a square at one end where you can write a label - go to the edge between the label area and the glass slide and focus on this line, then move the microscope stage back to where you stained the bacteria.

Once you've been able to focus on the plane of the slide, move up to 40X magnification and again check that your stained area is in focus. If not, adjust as needed, per above. It's important to not identify organisms as Gram positive or Gram negative at this magnification, as interpretation of the stain can be misleading. Always make your final interpretation at 100X magnification.

At this point you'll need to add immersion oil to view bacteria under 100X. A drop over the area to be viewed is more than enough. Once immersion oil is added *never* go back to a 40X or 20X "high and dry" objective as oil will get on the lenses, rendering them useless. To refocus, use a 4X or 10X objective, being very careful not to touch the oil.

Interpretation:
Once you have the slide in focus at 100X, here are some things to evaluate:

How good was your Gram stain? In real life we perform something called "Quality Control" to make certain the Gram stain worked as it should. We test all reagents against a known Gram negative species (E. coli) and a known Gram positive species (Staphylococcus aureus). If these organisms don't give a clear red/purple stain, then the staining method needs to be altered, or we use new reagents. If you don't have known microbial species available, try Gram staining the organisms in your mouth. Gram stain of your oral flora will show both Gram positive and negative species. If you see only one or the other, then you're stain wan't performed properly, or there was a problem with the reagents. If everything is Gram positive - try a longer decolorization step. If everything is Gram negative, try a shorter decolorization step.
Are stained cells Gram positive or Gram negative? If your Gram stain appears adequate, evaluate whether the organisms you see are Gram positive (purple) or negative (red). Be certain to observe many fields on the slide, preferably in areas where the organisms are not clumped together. Dense areas will often stain purple, even with Gram negative species.
What is the size of the cells you are viewing? Microbes are very small, frequently 10 nanmeters or less (0.000000010 meters!). Debris on the slide will be much bigger. Likewise eukaryotic cells, such as cells in your body are much larger, frequently 10 times or more. If you stained your cheek, you'll notice shed cheek cells which have a fried-egg appearance, where the cell cytoplasm is faint pink (and shaped like the white of the egg), while the nucleus is darker - often light purple (shaped like the yolk). These cells are often >10 microns in diameter. Bacteria often stick to cheek cells, so you may be able to find both Gram positive and Gram negative species stuck to a cheek cell. Fungi and molds will stain Gram positive as well - look for large filaments that are of greater width than bacteria, or "Schmoo cells" which are likely yeast cells.
What is the morphology of the bacteria? Round organisms are called cocci. Oblong organisms are called rods or bacilli. Slightly oval bacterial are called "coccobacilli". Bacterial cells can also be filamentous, just like fungi, but the filaments are not as wide. Many soil bacteria will produce filamentous forms. You may also find bacterial cells that have areas of red and purple in them - if you have an adequate Gram stain, these organisms would be called "Gram variable." Species of Clostridia and other organisms are frequently Gram variable - the lack of a definitive Gram stain occurs due to alterations in the lipds in their cell wall, which repels some of the stains.