The Gram Stain Preparation

The Kingdom_Prokaryotae (formerly Kingdom_Monera) includes the Archaebacteria (ancient forms) as well as the Eubacteria (modern bacteria). When bacteria were first discovered, the available technology revealed that all known forms had cell walls composed of a carbohydrate substance called peptidoglycan. Over time, new technology provided the means to discover that some very small Eubacteria lack cell walls and also that some of the Archaebacteria either lack cell walls or have cell walls that are made of substances other than peptidoglycan.

Christian_Gram’s bacterial cell wall staining process has been used since the 1800s for placing all bacteria that have cell walls made of peptidoglycan into two classification groups within their kingdom, based on the cell's response to his staining procedure: Gram_positive (Gram+) = purple color and Gram_negative (Gram-) = red/pink color.

Bacteria that are Gram+ have a cell wall structure with layers of peptidoglycan lying above their cell membrane. The layers are secured by amino acids. Gram- bacteria have cell walls that are stacked with a membrane layer, a layer of peptidoglycan and a top membrane layer.

Because of the differences in their cell wall structure, the Gram+ and Gram- bacteria will respond differently not only to Gram’s staining process, but also to various antibiotics. Hence, the Gram stain is often used in diagnosis. The exact type of bacteria causing an infection should be identified before physicians prescribe antibiotics. Gram+ and Gram- organisms will not respond to an antibiotic that cannot interact with their surface structure.

Staining provides contrast between microorganisms and their background, permitting differentiation among various morphological types. It also permits the study of the internal structures of the bacterial cells by providing better contrast at higher magnifications. In order to make a bacterial slide you must recall aseptic_technique.

PROCEDURE

(1) Obtain a clean microslide, Bunsen_burner, striker, inoculation_loop and either a tube of mixed liquid bacterial_culture or a Petri_dish with colonies.

(2) If you are using the liquid culture, you will flame the open tube, flame the loop, cool the loop and use the loop to transfer a loop of culture solution onto the center of the slide. Keep the amount of culture small. A large drop of culture solution will pick up so much stain that you will not be able to observe individual cells.

(3) If you have a prepared Petri dish of culture instead of a tube of liquid culture, you will have to place a drop of water on your microslide and then use aseptic technique to transfer a small amount of one colony from the Petri dish into the drop of water.

(4) Slides must air_dry before continuing the procedure. The organisms will form a film on the surface of the slide.

(5) Once the film has dried the slide must be heat_fixed so that the organisms do not wash off the slide during the remainder of the staining procedure. Use a clamp or spring-action clothes pin to hold the end of the slide, pass the slide just above the tip of the Bunsen burner flame three times - film side up.

CAUTION: stay just above the top of the flame because exposure to intense heat will distort the shape and structure of the bacterial cells.

(6) Hold the slide in the clamp for several minutes while it cools.

(7) Place the slide on the staining_rack located near the large sinks at the ends of our lab tables.

(8) Add Crystal_Violet stain over the surface of the slide and leave it there for 30 seconds.

(9) Rinse the slide with a dropper bottle of water – flood it over the surface of the slide.

(10) Cover the slide with Grams_iodine solution (this is a mordant: a substance that makes the cell more receptive to a stain) and leave it on for 30 seconds.

(11) Rinse with water.

(12) Decolorize the slide with 95% alcohol to remove excess stain from the cells. If the film is thin leave the alcohol on for 12-20 seconds. If the film is thicker, leave the alcohol on until the alcohol drops that are rolling off the slide no longer have color (add more alcohol as needed).

(13) Rinse with water.

(14) Counterstain with Safranin for 20-30 seconds.

(15) Rinse with water and blot dry.

(16) Examine under the oil_immersion lens.

Bacteria that are Gram+ will be able to retain the purple Crystal violet color even after they have been decolorized. Gram- bacteria will lose their purple color after decolorization, but will retain the red/pink Safranin counterstain. The reason for this difference in color retention relates to the structural differences in the cell walls of the Gram+ and Gram- forms.