Spring, 2024
North South University
Department of pharmaceutical Sciences
Course: PHR124L (Pharmaceutical Microbiology-I Laboratory)
[Exp-03] Title of the Experiment: Preparation of bacterial Smear from both a liquid broth
and agar medium followed by simple staining and microscopic observation.
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Objective: The purpose of simple staining is to elucidate the morphology and arrangement of
bacterial cells. staining is also called as monochrome staining or positive staining. 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. Examples of simple stain are Methylene blue, Safranin, Malachite green, Basic
fuchsin and
Materials required:
• Clean microscope slides
• Staining trays and newspaper
• Water bottle (for rinsing)
• Dropper bottle containing staining solution of methylene blue
• Brightfield Light Microscope
• Oil immersion
• lens paper
• bibulous paper
• Bacterial cultures: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa
Preperation of Bacterial Smear
1. Label a clean glass slide using a red wax marker.
2. Add a small drop of saline or distilled water to the slide.
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.
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.
6.After heat-fixing is complete, you are ready to simple stain your slide.
Page 1 of 3
Spring, 2024
North South University
Department of pharmaceutical Sciences
Course: PHR124L (Pharmaceutical Microbiology-I Laboratory)
[Exp-03] Title of the Experiment: Preparation of bacterial Smear from both a liquid broth
and agar medium followed by simple staining and microscopic observation.
__________________________________________________________________________________
Staining procedure by methylene blue or any primary stain:
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.
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.
Precautions:
• Be careful not to overheat the slides in this procedure
• Avoid getting stain on your clothes, books, and fingers. It is very difficult to
remove.
• Hold your slide diagonally while rinsing your extra dye.
Page 2 of 3
Spring, 2024
North South University
Department of pharmaceutical Sciences
Course: PHR124L (Pharmaceutical Microbiology-I Laboratory)
[Exp-03] Title of the Experiment: Preparation of bacterial Smear from both a liquid broth
and agar medium followed by simple staining and microscopic observation.
__________________________________________________________________________________
Page 3 of 3
