An Eco-friendly, Scaled-down Gram Stain Protocol

An Eco-friendly, Scaled-down Gram Stain Protocol

Ruth A. Gyure *
Western Connecticut State University, Danbury, CT 06810.

INTRODUCTION

The Gram stain protocol is perhaps one of the most commonly used methods in microbiology and is taught in just about every introductory microbiology lab. Many instructors have been doing it the same way that they were taught, using a staining rack held over a sink or other large receptacle and “flooding” the slides with large amounts of stain or reagent (easily several liters per student) during each step.

Currently, flushing large volumes of Gram stain reagents into sanitary sewage systems is no longer acceptable. These chemical wastes are highly regulated and must be collected, labeled, and disposed of in a responsible manner, usually by paying a commercial service to remove them to an authorized off-site facility. Such services are costly and, as expected, costs are proportional to volume of collected waste. This “old” method of Gram staining, even if effluent is collected, generates a high volume of liquid waste which is unnecessarily diluted with additional large volumes of water from the rinsing steps.

The purpose of using this scaled-down and eco-friendly protocol is to dramatically reduce the amount of liquid waste produced without sacrificing quality of results. This protocol is flexible, practical, and easy to implement. It does not require students to work at a bench sink, reduces user cost, and lowers environmental impact overall.

SET-UP

Figure 1 shows a typical setup, one that the author has used for several years. It consists of:

 


 

FIGURE 1 .  The station set-up for this protocol. Two alternative containers for collecting stain are shown. On the left is a glass culture dish (easier to keep clean); on the right is a typical disposable plastic container which is very cost-effective and easy to replace when it gets too unsightly or cracked.

  1. A washable mat to keep work area neat. Low cost suggestions include plastic cutting boards available at grocery and discount stores, often in bulk packs.

  2. A glass culture dish (six-inch diameter, two-inch depth) to hold stain/reagent effuent. An alternate receptacle is also shown here.

  3. Anodized aluminum mesh screen cut into six-inch-by-six-inch squares. The official mesh specifications are #4 Mesh, openings 0.20 inches. Other opening sizes will do, but aluminum is a must to prevent rusting. This material can be bought by the yard at hardware stores for a few dollars. It cuts easily with a good sturdy pair of regular scissors.

Other than this difference in setup, other materials and reagents are the same (squirt bottles of staining reagents, water for rinsing steps, tissues or other paper for blotting).

PROTOCOL

In general, the steps and timing of the Gram stain are the same. One starts with a heat-fixed smear. Next, instead of flooding the slides with crystal violet, iodine, or safranin, one simply uses a small “puddle” of stain on top of the heat-fixed smear as follows:

  1. Apply a small amount of Gram’s crystal violet on top of the heat-fixed smear, letting the slide sit flat on the wire mesh screen over the effluent bowl. This is a tiny volume (one to two milliliters) and should form a convex puddle that is not running over the sides of the slide (Fig. 2).

  2. After about 20 seconds, student should lift slide at an angle and drain off this small amount of stain. Lay it flat again and repeat Step 1, letting stain sit another 20 seconds. Drain off the stain, lay flat and repeat Step 1 once more. Stain should sit on the side for a total of about 60 seconds, with these two intervals of draining and refreshing in between.

  3. Proceed with water rinse, then thoroughly dry the slide/smear. Students can be taught to be frugal in the use of water. This is the step where most waste volume will accumulate if students are careless and needlessly pour large amounts on the slide.

Once slide has been rinsed and dried, repeat Steps 1–3 with Gram’s iodine. Proceed to destaining step. Finally, repeat Steps 1–3 with Gram’s safranin.

 


 

FIGURE 2 .  This photograph shows the amount of stain that should be “puddled” on top of the smear. Note that it should not run over the sides. Also, the clothespin helps to hold slide when moving it, but should be removed during staining so that the slide lays flat on the screen.

FLEXIBLE MODIFICATIONS OF SETUP AND PROTOCOL

Instead of plastic cutting board sheets, other users have reported scavenging old cafeteria trays, placemats, or other materials that work equally well. The important thing is durability and washability. The tray’s purpose is to keep bench and work area clean and neat. Containers other than glass culture dishes can be used to collect effluent. Reusable plastic-ware, aluminum trays, or other receptacles would be adequate. Glass dishes have the advantage of cleaning up very thoroughly and not accumulating stain. Finally, aluminum mesh is sturdy, washable, rust-resistant and provides a stable platform for slides during staining. But others have used wooden sticks or skewers taped together at a workable distance to hold slides over the dish. Resourceful educators will come up with many more such suggestions!

SAFETY

When the procedure is done, all liquid effluent must be collected in a labeled container (Fig. 3) and disposed of according to regulatory guidelines for each institution and state. There are no additional considerations for this protocol above those used in doing any Gram staining procedure. Goggles are required for working with liquids in the laboratory and gloves are highly recommended. Students should be trained in appropriate microbiological techniques for handling live cultures when preparing their heat-fixed smears.

 


 

FIGURE 3 .  Waste from the collecting dishes must be placed in an appropriate labeled container for hazardous waste removal. At many institutions, it is acceptable to collect waste in used, washed containers such as this old bleach bottle. Alternatively, special containers may be provided by your institution. In either case, careful labeling according to state and local regulations is essential.

*Corresponding author. Mailing address: Department of Biological and Environmental Sciences, Western Connecticut State University, 181 White St., Danbury, CT 06810. Phone: (203) 837-8796. Fax: (203) 837-8769. E-mail: gyurer@wcsu.edu .

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DOI: 10.1128/jmbe.v11i1.144
Journal of Microbiology & Biology Education , May 2010
Copyright © 2010 American Society for Microbiology . All Rights Reserved



JMBE
ISSN: 1935-7885