Chapter 11 Lab Exercises
Section 9 Flow Through Gram Stain
Page 2 Instructor

Flow Through Gram Stain

Instructor Version (go to Student Version)

Subject Area(s) microbiology
Intended Audience
high school biology, independent study/science fair, introductory undergraduate microbiology, advanced college level microbiology
Type laboratory exercise
Revision Date May 12, 2008


The Gram stain devised by Christian Gram in 1882 has become one of the most important diagnostic procedures in microbiology. This stain differentiates two main categories of bacteria according to the structure of the cell wall. Bacteria with a predominantly peptidoglycan cell wall stain blue/purple with this technique and since they retain the primary stain are called Gram positive (Gram +). Cells with only a minor peptidoglycan component and possessing a lipopolysaccharide cell wall give up the primary stain during destaining and therefore exhibit the red counter stain. These cells are called Gram negative (Gram -).

A Gram stain is usually performed on a smear preparation that has been heat fixed. One function of fixation is to secure (fix) the cells to the slide. In a biofilm, however, the cells are already firmly attached. Furthermore, a heat fixed slide is dry, but a biofilm is mostly water. Drying alters the biofilm virtually beyond recognition. This exercise describes a method for obtaining a Gram stain on a minimally altered biofilm.


Students should be able to define a biofilm, describe the differences between biofilm (surface-attached) and planktonic (free-floating) bacteria, and be able to describe why bacteria usually grow on surfaces. Students should also be familiar with the standard method of preparing a Gram stain and should understand the mechanism of this procedure.


Given readily available materials, detailed instructions and a biofilm preparation on a 1 x 3 inch microscope slide the student will be able to prepare a Gram stain which does not require heat fixing or drying the preparation.


Preparing the biofilm for staining

The student may prepare a biofilm slide for Gram staining by any practical method.
  1. They could use one of the methods described in Biofilms: The Hypertextbook.
  2. Perhaps the simplest method is to simply locate a clean 1 x 3 inch microscope slide at some site likely to produce a biofilm, a stream, pond, seep, wharf, shower, drain or aquarium. Any site where a slide can be submerged in water will serve. Flowing water is preferable as movement of water produces a more robust and tightly attached biofilm.

Detailed instructions for the preparation of the slide for Gram staining and for the staining procedure itself are contained in the Student Attachment which can be reached by clicking on Detailed instructions and illustration for flow through Gram staining listed under Attachments.

Teachers Note: The thickness of the preparation may preclude an examination under oil immersion. This procedure gives an overall view of the biofilm with microcolonies, streamers, and water channels. Although resolution of individual cells may be difficult, the Gram Stain composition of the visible micro colonies can be determined.

Safety Note: It is recommended that students wear lab coats or an apron to protect clothes from staining and gloves to protect their hands. Students should wash their hands before leaving the lab since they are working with bacteria.


1 biofilm grown on a 1 x 3 inch microscope slide
1 25 mm square coverslip (the thinner the better)
As Necessary petroleum jelly
As Necessary paper towels
1 Gram Stain Kit (Hucker’s Crystal violet, Gram’s Iodine, Decolorizer, Safranin)


The instructor may assess the exercise by examining the student prepared biofilm Gram stains. Students should have the opportunity to examine and compare the Gram stained biofilms of other students with that they have prepared.


This exercise may be linked with any method of producing a biofilm, including the Batch Biofilm Growth Reactor, the Continuous Flow Stirred Reactor, the Static Glass Coupon Reactor, the Drip Flow Reactor, the Henrici’s Microbe Capture Technique, or the Buried Slide Technique. Alternatively, sterile glass microscope slides can be sited in streams, ponds, seeps, drains, etc to produce a “natural” biofilm.


Brown, Alfred E. 2008.

Benson's Microbiological Applications: Laboratory Manual in General Microbiology, Short Version, McGraw-Hill.

This material is based upon work supported by the National Science Foundation under Grant No. 0618744, and in part by the Waksman Foundation for Microbiology. Developed in collaboration with Dr. John Lennox, Penn State Altoona. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.

©2002-2008 Center for Biofilm Engineering,