Columbia University

Summer Research Program for Science Teachers

Manhattan Center for Science and Mathematics

Sau Ling Chan

1997

Objective

Background Information for Teachers

What Does My Bacteria Look Like?

Purpose of Activity: To introduce Gram’s Stain as a standard practice for bacterial identification.

Type of Activity: Hands-on activity

Group/Cooperative Learning

Review/Reinforcement

Type of Skills Emphasized: Good observation

Analytical/Reasoning Skills

Basic Research Techniques

Aseptic Techniques

Target Audience: Microbiology

Biology

Abstract of Activity: The following activity incorporates a number of laboratory techniques, including the use of microscope, Gram’s Stain, and a basic understanding of bacteria morphology. [Content Standard Unifying Concepts- Systems, order, and organization] Students can work in groups of two to find out the name and type of bacteria that they are given to study. However, a brief discussion should be held before this experiment regarding the function and application of Gram’s stain. Prior experience with Gram’s stain on known bacteria is highly recommended.

It is extremely important to remind students of aseptic techniques when working with dangerous chemicals and potentially infectious microorganisms. To ensure that students know what to do, it would be helpful for the teacher to perform a demonstration of Gram’s stain. [Teaching Standard D- Ensure a safe working environment] Depending upon the science background of students with whom you work, the biochemistry behind this method could be explained as an enrichment activity of this lab.

Objective of Activity: To identify an unknown microorganism using good observation and logical reasoning skills through an inquiry-based approach. [Teaching Standard A- Select and adapt content to meet students' abilitities]

About Gram’s Stain: Gram’s Stain is a double-staining method which forms the basis of most examination and the preliminary identification of bacteria. Differential staining involves the use of several stains. The technique shows the differences between different bacteria and different cell structures.

The Gram stain allows us to divide bacteria in to two groups, Gram positive and Gram negative. The Gram positive bacteria appear violet or blue in color, and the Gram negative appear red to pink in color. These two types of are different in their susceptibility to drugs. By identifying bacteria as positive or negative, a diagnosis and treatment can be made easier.(Edwards, 1994) [9-12 Content Standard E- Understandings about science and technology]

Please note that the test should be carried out on young cultures, within 18-24 hours, since some bacteria change their Gram reactions as the cultures age. Although there are many modifications of Gram’s stain, the method introduced here are particularly suitable for class and routine use.(Harrigan, 1966)

Preparation of Gram’s Stain Reagents:

Crystal violet solution

Crystal violet 0.5 g

Distilled water 100 ml

Gram’s iodine solution

Iodine 1.0 g

Potassium iodide 2.0 g

Distilled water 300 ml

Dilute carbol fuchsin solution

Basic fuchsin 1.0 g

Absolute ethanol 10 ml

Phenol 5.0 g

Distilled water 1600 ml

This may be more conveniently prepared by diluting one part of Ziehl-Neelsen’s carbol

fuchsin in 15 parts of water. (Harrigan, 1966) [Teaching Standard D- Make accessible science tools]

LESSON OVERVIEW

I. Introduction

Brief discussion of the two main types of bacteria—Gram + vs. Gram -.

Discuss the difference between Gram + and Gram - bacteria. Give one example of each.

How does knowing whether a bacteria is Gram + or Gram - help researchers design an appropriate experiment for study?

What are the three common morphology found in bacteria? Give one example of each. [Teaching Standard B- Orchestrate scientific discourse]

II. Identifying Bacteria

Give each pair of students a pure culture of unknown strain. They should be able to deduce the type of bacteria based on Gram’s reaction, and its morphology by examining the shape of single bacteria along with the texture of the colony. To make things easier, provide a list of possible bacterial strains. They may check their preliminary guess against the bacteria chart and prepared slides provided to them.

It is best to check that students have successfully identified the unknown before researching the type of diseases related to the strain given to them. Otherwise, that would defeat the purpose of the entire lab. For instance, if two similar strains were tested in this lab, completing the research aspect first could easily mislead students to draw the wrong conclusions. If necessary, students may use notes taken in prior labs to aid identification.

III. Varying the Level of Difficulty

The level of difficulty of this lab is variable. Teachers can provide cultures of bacteria that students already learned in previous labs or select a closely-related strain that belongs to the same family that was never introduced before. Other characteristics, such as performing a motility test with positive and negative controls, can be used as extensions of the lab.

Source: Jawetz et al. [9-12 Content Standard C- Organization in living systems]

References: Edwards, G.I. et Cimmino, M. Laboratory Techniques for High Schools. Barrons.

New York. 1994.

Harrigan, W.F. et Mc Cance M.E. Laboratory Methods in Microbiology.(Fourth

Edition) Academic Press. New York. 1966.

Jawetz, E., Melnick, J.L., and Adelberg, E.A. Review of Medical

Microbiology.(Seventeenth Edition) Appleton & Lange. Connecticut. 1987. [Teaching Standard D- Make accessible science tools]


Worksheet for Students

. What Does My Bacteria Look Like?

Name of Students: 1. ________________________ Date: _________________

2. ________________________

Unknown # ___

Materials/Equipments Needed: Compound Microscope Bacteria Chart

Gram’s Stain Protocol Glass Slides

Cover slips Microbiology/Medical texts

Sterile Cotton Swabs Staining Tray

Prepared Slides of Various Bacteria

Chemicals Needs: Crystal Violet Solution Distilled water

Gram’s Iodine Solution 95% Ethyl Alcohol

Carbol Fuchsin Solution Safrarin

Procedures: 1. Obtain a culture of bacteria from your instructor.

Using sterile cotton swabs, make a thin smear of the bacteria onto glass slide.

Flood the fixed smear with Gram’s crystal violet stain for 1 minutes. Be sure the slide is on the staining tray.

Pour off the dye and wash slide with water.

Flood the slide with Gram’s iodine for one minute. Iodine is a mordant, which fixes the Gram + cells so that they will not lose the violet color.

Pour off the iodine and wash. Decolorize the slide with 95% ethyl alcohol. Add the alcohol a drop at a time until the material running off the slide is colorless.

Remove the alcohol at once by washing in cold water.

Flood the slide with safranin for 1 minutes.

Pour off safrarin, wash slide, blot dry.

Examine slide. Complete chart.

Note: If the bacteria are blue or violet, they are Gram +.

If the bacteria are red or pink, they are Gram -.

Check your answers against prepared slides and bacteria chart. Be sure to check your answers with the instructor before researching the possible type of diseases related to your bacteria.

Conclusion: Answer the following questions in paragraph form.

What are the three general morphologies of bacteria?

How did you identify the unknown bacteria? What evidence do you have to support your preliminary guess?

How does the structure of an organism relate to bodily functions? [Content Standard Unifying Concepts- Form and function]

Give two reasons why it is important to test whether bacteria is Gram + or Gram -? How do you suppose this would help biomedical research?

Extension: Perform a motility assay and answer the following questions for extra credit.

What advantage has differential staining over simple staining?

What is a mordant?

What is a counterstain?

Why is a decolorizer used?

Source: Edwards, G.I. et Cimmino, M. Laboratory Techniques for High Schools. Barrons. New York. 1994.


Information for Future Experiments

Gram’s Stain

About Gram’s Stain: Gram’s Stain is a double-staining method which forms the basis of most examination and the preliminary identification of bacteria. Differential staining involves the use of several stains. The technique shows the differences between different bacteria and different cell structures.

The Gram stain allows us to divide bacteria in to two groups, Gram positive and Gram negative. The Gram positive bacteria appear violet or blue in color, and the Gram negative appear red to pink in color. These two types of are different in their susceptibility to drugs. By identifying bacteria as positive or negative, a diagnosis and treatment can be made easier.(Edwards, 1994)

Please note that the test should be carried out on young cultures, within 18-24 hours, since some bacteria change their Gram reactions as the cultures age. Although there are many modifications of Gram’s stain, the method introduced here are particularly suitable for class and routine use.(Harrigan, 1966)

Preparation of Reagents:

Crystal violet solution

Crystal violet 0.5 g

Distilled water 100 ml

Gram’s iodine solution

Iodine 1.0 g

Potassium iodide 2.0 g

Distilled water 300 ml

Dilute carbol fuchsin solution

Basic fuchsin 1.0 g

Absolute ethanol 10 ml

Phenol 5.0 g

Distilled water 1600 ml

This may be more conveniently prepared by diluting one part of Ziehl-Neelsen’s carbol fuchsin in 15 parts of water.

Source: Edwards, G.I. et Cimmino, M. Laboratory Techniques for High Schools. Barrons. New York. 1994.

Harrigan W.F., Mc Cance M.E. Laboratory Methods in Microbiology. Academic Press. New York. 1966.

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