A bacterial smear is a preparation where microorganisms are thinly spread and fixed onto a glass slide, forming the fundamental starting point for microscopic analysis. The primary purpose is to allow scientists to visualize bacteria, which are naturally transparent and nearly invisible under a standard light microscope. Staining the fixed smear enhances the contrast between the cells and the background, revealing details about the bacteria’s shape, size, and arrangement. This preparation is necessary for subsequent identification methods, particularly differential staining techniques that help broadly classify unknown species.
Preparing the Bacterial Smear
Smear preparation depends on the culture medium used. If the sample comes from a liquid broth culture, one or two loopfuls of the liquid are transferred directly onto the clean glass slide and spread evenly over a small area. When using bacteria grown on solid agar, a small drop of water is first placed on the slide, and a tiny amount of the bacterial colony is mixed in to create a slightly cloudy suspension. The smear must be thin so that individual cells are not clumped together, which is necessary for accurate viewing.
After spreading, the slide must air-dry completely at room temperature to remove excess water. The next step is fixation, typically achieved through the application of heat. Heat fixing involves quickly passing the underside of the dried slide through a flame a few times until the glass is warm to the touch. This process kills the bacteria and causes the microbial cells to adhere firmly to the glass surface. The heat coagulates the bacterial proteins, cementing them to the slide so they do not wash off during subsequent staining and rinsing steps.
Principles of Bacterial Staining
Staining is necessary because bacterial cells lack natural color and cannot be seen clearly against a bright background in a light microscope. The dyes used in microbiology are colored chemical compounds that work primarily through electrostatic attraction. Since most bacterial cell surfaces carry a net negative electrical charge, basic dyes—which have a positively charged colored component (cationic chromophore)—are readily attracted to and bind with the surface, staining the cell itself.
Staining techniques are divided into simple and differential staining. Simple staining uses only one dye, such as methylene blue or crystal violet, to color all cells uniformly, which is useful for quickly determining basic morphology and arrangement. Differential staining employs multiple reagents to distinguish between different types of bacteria based on their distinct chemical or physical properties. The Gram stain is a primary example of differential staining, classifying bacteria into two major groups based on their cell wall structure.
Step-by-Step Gram Staining Procedure
The Gram stain differentiates bacteria by exploiting differences in their cell wall composition, particularly the thickness of the peptidoglycan layer. The process begins with the application of the primary stain, crystal violet, to the heat-fixed smear, which stains all bacterial cells purple. The crystal violet dye is typically left on the slide for about 30 to 60 seconds before being rinsed off with water.
The second step involves flooding the smear with Gram’s iodine solution, which acts as a mordant. The iodine forms a large, insoluble complex with the crystal violet inside the bacterial cells. The excess iodine is rinsed away with water, leaving both Gram-positive and Gram-negative cells still appearing purple.
Decolorization is performed using a solvent like 95% ethanol or an acetone-alcohol mixture. In Gram-negative bacteria, this solvent dissolves the outer membrane and washes the crystal violet-iodine complex out of the thin peptidoglycan layer, leaving the cells colorless. Conversely, in Gram-positive bacteria, the thick peptidoglycan layer becomes dehydrated by the alcohol, shrinking and tightening its mesh-like structure to trap the large crystal violet-iodine complex inside, so the cells retain the purple color. The decolorizer is applied only until the runoff liquid appears clear, usually requiring only a few seconds, and then immediately rinsed with water to stop the process.
The final step is the application of a counterstain, typically safranin, which is a red or pink dye. This stain is absorbed by the colorless, decolorized Gram-negative cells, causing them to appear pink or red under the microscope. Gram-positive cells, which are already intensely purple from the initial stain, are not affected by the safranin and remain purple. The resulting color difference provides a fundamental tool for bacterial identification.
Troubleshooting and Interpretation
Accurate Gram stain results require attention to detail, as several common errors can lead to misinterpretation. Preparing a smear that is too thick prevents the decolorizer from penetrating evenly, potentially causing Gram-negative cells to incorrectly appear purple. Over-heating the slide during the heat-fixing step can also damage the bacterial cell walls, making Gram-positive organisms unable to retain the primary stain, thus yielding a false Gram-negative result.
Decolorization errors are common: under-decolorization leaves Gram-negative cells purple, leading to a false Gram-positive reading, while over-decolorization removes the stain from the Gram-positive cells, causing them to appear falsely Gram-negative. To view the final slide, immersion oil must be used with the highest power objective lens (100x) to enhance resolution by reducing light refraction. Interpretation involves observing the color to determine the Gram reaction, and noting the cell morphology and arrangement.