4.3: Lab Procedures- Bacterial Smear, Simple and Gram Staining

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Learning Outcomes

Prepare microorganisms for microscopic observation.
Observe the difference in size between bacteria and other unicellular microorganisms.
Perform a simple stain and a Gram stain.
Observe stained microorganisms and identify their size, shape, and staining properties.

Part I: Preparation of a Bacterial Smear

This semester you will be performing two staining procedures: A Simple Stain and a Gram stain. Both of these staining procedures begin with the preparation of a bacterial smear.

Materials

Clean microscope slides
Staining trays and newspaper
Water bottle (for rinsing)
Bacterial cultures: Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Pseudomonas aeruginosa,

How to make a Bacterial Smear

1. Label a clean glass slide using a red wax marker. Note that it is important to recognize the side of the glass slide that you put your bacterial sample on.

2. Add a small drop of saline to the slide (you will usually put two bacteria on one microscope slide- Follow your instructor’s specific instructions). This can be done by placing a drop of saline onto your inoculation loop and then transferring it to the slide. If you use the saline dropper directly on the slide, do not release a full drop.

3. With an inoculation loop or needle, pick up a small amount of bacteria. Mix it well with the saline and spread the mixture over a wider area of the slide.

Be careful not to have the two smears run into each other.

4. Air dry the bacterial specimen on the slide (slide warmers may also be used).

5. When slides are completely air-dry, heat fix the bacterial specimen by passing the slide slowly over the flame twice (your instructor will demonstrate this).

Heat fixing kills cells, and adheres them to the slide.
Cells will be rinsed off the slides if they are not heat fixed properly.
Be careful not to overheat the slides in this procedure

After heat-fixing is complete, you are ready to simple or gram stain your slide.

heat fixing.png

Image 1: Heat Fixing over a bunsen burner

Watch Video 1: on heat fixing using a bacticinerator

Watch Video 1: on heat fixing using a bacticinerator from Dr. G. Kaiser at URL: https://youtu.be/Rh0GrcTzjnU

Part II: Simple Stain

Cultures

Bacillus megaterium

Micrococcus luteus

Saccharomyces cerevisiae - a yeast

Materials

Dropper bottle containing staining solution of methylene blue

Brightfield Light Microscope

Oil immersion

lens paper

bibulous paper

Lab Procedures

A. Review Lab procedures for operating a Brightfield Light microscope

B. Preparation of a Bacterial Smear for Staining

Preparations of bacteria for staining can be made from growth on an agar plate or from a broth culture.

1. To prepare a slide from cells grown on an agar medium, first place a SMALL drop, a loopful works well, of water on a clean grease-free slide. Next, with a sterile loop transfer a SMALL AMOUNT of the growth to the drop of water and rub the loop around until the material is as evenly distributed as possible to form a just visibly turbid suspension. Spread the drop over a small portion of the slide to make a thin film with lightly visible turbidity.

2. Prepare three separate smears, one each of Bacillus megaterium. Micrococcus luteus, and Saccharomyces cerevisiae.

3. Next, heat fix the slide by placing it on the slide warmer for 5 minutes. Put the sample side facing up and label with your initials on one end of the slide. This process is called heat fixing the specimen to the slide. Its purpose is to bind the specimen to the slide so that it does not wash off during staining. Killing the cells with heat fixation also increases their permeability to the dyes used in staining.

Do not under-fix (the smear will wash off) nor over-heat (the cells will be ruptured or distorted) your specimen. The slide should be warm to the touch, not hot. If you think you have heated the slide too much, do not touch the slide to your hand to find out. Chances are, your suspicions are correct, and you will burn your hand with the hot glass. Instead, heat-fix the slide for a shorter period of time next time, then test.

4. After cooling the specimen is ready for the simple stain.

C. Methylene Blue – A Simple Stain

Living bacteria are almost colorless, and do not present sufficient contrast with the water in which they are suspended to be clearly visible. The purpose of staining is to increase the contrast between the organisms and the background so that they are more readily seen in the light microscope. In a simple stain, a bacterial smear is stained with a solution of a single dye that stains all cells the same color without differentiation of cell types or structures. The single dye used here is methylene blue, a basic stain. Basic stains, having a positive charge, bind strongly to negatively charged cell components such as bacterial nucleic acids and cell walls.

1. Stain by covering the smear completely with methylene blue. This should be
done over a sink with a slide holder.

*Avoid getting stain on your clothes, books, and fingers. It is very difficult to remove.

2. Allow the stain to act 1-2 minutes.

3. Tilt the slide to allow the stain to drain off. Now, rinse the remaining dye off with a gentle stream of water from a faucet or wash bottle.

4. Blot the slide dry with bibulous paper. Remove excess water from the slide by touching one corner of the slide to the blotting paper, then place the slide between clean sheets of paper in the blotting pad and blot dry. Be sure you do not rub off the smear.

5. Examine under the microscope using first the 10X and then the 100X oil-immersion objective.

6. Record your observations on the report sheets.

D. Test plate isolate

1. Check your "test plates" from Lab1: Exercise I, part D (ubiquity of microorganisms) for isolated single colonies to be candidates for your test plate isolate.

2. Attempt a simple stain of your candidate test plate isolate as described in part B of Exercise II. Record your observations.

3. Show an instructor your isolated single test plate isolate and the slide of the simple stain of the specimen you have chosen. This is to ensure you will be working with a bacterial culture instead of a fungal isolate.

4. If you did not obtain any single colony bacterial isolates on your "test plates" use one from another student in the lab, but make sure you choose a different isolate than that student has chosen, or use one from the instructor's plates.

5. After picking a candidate for your test plate isolate it is necessary to make sure it is a pure culture. Use a new Nutrient agar plate to re-streak for single colonies using the technique from Exercise I, part C (the "T" streak). Make certain you try and touch only one colony, the one you are interested in using as your unknown. Save your original plate. Incubate until the next laboratory period.

E. Demonstration Slide

You have just prepared slides of Bacillus (a rod shape) and Micrococcus (a coccus). We have set up a slide to demonstrate a third major cellular shape seen in bacteria. Spirillum volutans is a spiral shaped microorganism found in fresh water. View the demonstration slide and record your observations in the Results page.

Part III: Gram Staining

Gram Staining Procedure

For all steps in the gram staining procedure, add enough of the solution to cover the areas of the slide that have bacteria on them. You do not need to flood the entire slide.
All staining should be done over a staining tray. Be sure to put newspaper under the tray in case of spillage.
Gloves should be worn while staining and removed before working with the microscope.

STEPS	EXPLANATION
1. Add a few drops of crystal violet (primary stain) to the smear and let it sit for 60 seconds.
2. Rinse the slide with water.	All cells are purple after this step. Stopping here would be a simple stain.
3. Add a few drops of Gram's iodine (mordant) to the smear and let it sit for 60 seconds.	Gram’s iodine forms a complex with crystal violet
4. Rinse the slide with water.	All cells are purple after this step.
5. Decolorize with 95% ethanol: let the alcohol run over surface of slide until no more crystal violet color comes out of the smear (time varies—no more than 5-10 seconds).	Gram positive cells retain crystal violet and remain purple. Gram negative cells lose crystal violet and are now colorless
6. Rinse with water.	Water rinse stops the decolorization process
7. Add a few drops of safranin (counterstain) and let it sit for 60 seconds.	Safranin is a pink/red dye
8. Rinse with water. Blot dry on bibulous paper. Be careful not to wipe off the bacteria.	Gram positive cells remain purple; gram negative cells are now pink/red
9. Observe your slide under the microscope.	For each organism, determine morphology, arrangement and Gram reaction

Watch Video 2: on Gram Staining

Watch Video 2: on Gram Staining. Video filmed at NC State. URL https://youtu.be/H-fxk1be1hQ