Henrici’s Microbial Capture Technique¹

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	April 15, 2008

INTRODUCTION

“Some time ago an aquarium in my laboratory developed on its walls a growth of algae. Some microscope slides were placed in the aquarium in the expectation that the algae would also grow on these and that permanent preparations might be made showing the organisms in situ. On removing and staining these slides after a week's immersion, I was surprised to find, in addition to the algae, a thin and uniform coating of bacteria of various forms, some of unusual morphology”. … “It is quite evident that for the most part the water bacteria are not free floating organisms, but grow upon submerged surfaces; they are of the benthos rather than the plankton”. Arthur T. Henrici

These comments written in 1933 indicate, that long before the term biofim was used², Henrici understood the basics of biofilm formation. Elsewhere in that same article Henrici showed that he had an understanding of biofilms that sounds modern even by contemporary standards. This understanding included the facts that 1) the bacterial cells are actually growing on the slide as confirmed by the presence of microcolonies, 2) that they are firmly attached to the glass slide, 3) that the cells are surrounded by a matrix (sheath of gum) which holds the cells to the slide 4) that specimens from aquaria and lakes alike produced these biofilms, 5) that most bacteria are not in the water column but rather are surface attached.

Using techniques very much like those described by Henrici it is possible to “fish” for bacteria, algae, protozoa and microinvertebrates by placing clean glass microscope slides in an aquatic environment such as aquaria, lakes, streams, showers, the tanks of toilet bowls, and any other site where there is a large or small body of water either standing or flowing.

PREREQUISITES

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 the growth of bacteria on surfaces. Students should also be familiar with basic microscopic techniques including brightfield microscopy and staining techniques. Access to a phase microscope would be useful for observing living unstained cells.

INSTRUCTIONAL OBJECTIVE

Given readily available materials and detailed instructions, students will be able to construct devices that will permit them to collect populations of surface attached bacteria, algae, fungi, living protozoa and microinvertebrates for study under a microscope. Using additional techniques found elsewhere in this collection, students may harvest and enumerate the bacteria by standard dilution and plating techniques or by using the drop plate method. See other lab exercises: Harvesting and Dispersing of Cells from Biofilms (Standard Method), Drop Plate Method for Counting Biofilm Cells

INSTRUCTIONAL PROCEDURES

1. Students will be provided with the materials required to grow biofilm on microscope slides in an aquatic environment, and given detailed instructions and diagrams for carrying out this procedure.
2. Students will be provided with appropriate staining kits (Gram stain, spore stain).
3. Students will be given an opportunity to review the materials and instructions and to ask questions concerning the procedure.
4. Detailed instructions for making this device are included in the student instructions.

Teacher Notes:

An aquarium, a hay infusion, an illuminated pond water sample, or one of the River Tanks distributed by Carolina Biological Supply Company makes an excellent classroom location for placing Micro-Fishing slides.

This procedure may be linked to other exercises including harvesting and dispersal of cells, dilution and enumeration, and drop plate counting. (See these exercises elsewhere in this collection).

The slides thus prepared may be stained with any of a wide variety of readily available dyes. Keep in mind, however, that biofilms are approximately 98% water and that heat fixing dramatically alters the appearance of this micro community.

The Flow Through Gram stain procedure, found elsewhere in this collection is a procedure for staining a minimally altered biofilm.

A multiple well slide can also be used in a microbial fishing exercise by masking all but one of the wells with transparent tape or Parafilm® M prior to placing the slide at its site. Then each day or at another selected interval, one additional well can be exposed. At the end of the sampling period each student has a sequence representing the growth of a biofilm over a period of time all on one slide.

Caution: If the slides are going to be sited in a natural area like a stream or pond it is a good idea to label them as a student experiment and place them out of sight. I have had student projects disturbed or intentionally ruined by curious or mischievous passersby.

MATERIALS AND EQUIPMENT

Quantity	Description
As Necessary	18 gauge wire
1	wire cutters and pliers and shears
1	a spine from a student report binder
As Necessary	rubber bands
As Necessary	microscope slides (or preprinted slides from Erie Scientific or other distributer)
As Necessary	transparent tape or Parafilm® M

ASSESSMENT / EVALUATION

Instructors can evaluate the success of this exercise by examining and discussing slides with the student.

FOLLOW-UP ACTIVITIES

This exercise may be included as part of a semester-long biofilm project called “Bring “Em” Back Alive” in which students isolate a biofilm from nature and characterize it over a semester. One step in this exercise asks the students to isolate one of the component strains from a biofilm, and demonstrate that it produces a biofilm by the colorimetric method described elsewhere in this series. Following the verification that the isolate is a biofilm producer the students could produce a growth curve according to the instructions given in “A Biofilm Growth Curve.”

REFERENCES

¹Studies of Freshwater Bactria: I. A Direct Microscopic Technique
Henrici, AT
Journal of Bacteriology 1933; 25:277-286

²How Bacteria Stick
Costerton JW, Geesey GG, Cheng K-J
Scientific American 1978; 238(1):86-95

³Flow induced vibrations, drag force, and pressure drop in conduits covered with biofilm
Lewandowski Z, Stoodley P
1995. International IAWQ Conference Workshop on Biofilm Structure, Growth, and Dynamics, 30 Aug. to 1 Sept., Noordwijkerhout, The Netherlands.

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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, http://www.biofilm.montana.edu