Bright-Field Light Microscope: Smear Preparation and Simple Staining
Abstract
Bacteria has three basic shapes which are cocci, rods, and spirilla. Through using the
bright-field light microscope, the three bacterial shapes and arrangement of prokaryotic
structure can be observed. The bright-field light microscope is an instrument that
magnifies images using two lens system. To observe the bacteria clearly, simple staining
will occur. In this experiment, four bacteria were stained and observed; Bacillus
megaterium, Saccharomyces, Escherichia coli, and Staphylococcus aureus.
Introduction
Due to bacteria’s lack of color and size, smear preparation and simple staining is
necessary. This is necessary because it allows the bacterial morphology to be observed. A
dried preparation of bacterial cells on a glass slide is a bacterial smear. Once a bacterial
smear is done correctly, the bacteria are evenly spread out on the slide yet separated from
one another, the bacteria are not washed o the slide during staining and the bacterial
form is not distorted. Then the bacteria can heat-fixed and killed in this way without serious
distortion of cell structure. A single stain or dye called a chromogen is used to create a
contrast between the bacteria and the background is referred to as simple staining. In this
experiment the dye that is used was methylene blue to create the contrast. The methylene
blue dye bind to bacterial cells by ionic interactions. The dye contains positively charged
groups and bind to negatively charged molecules like any proteins and the surfaces of
prokaryotic cells.
Procedure
1. Disinfect work area before and after you start the experiment.
2. Sterilize the inoculating loop and put a drop of water (distilled) on the slide using the
inoculating loop.
3. Sterilize the loop again and collect a small sample of yeast, making sure to barely touch
the agar disc.
4. Smear the yeast in the water that was placed on the slide. Make sure that it is a thin
smear and not milky. Let it air dry.
5. Burn the loop again.
6. Heat the slide 4-5 times until the yeast sticks to the slide (heat fix).
Abstract
Bacteria has three basic shapes which are cocci, rods, and spirilla. Through using the
bright-field light microscope, the three bacterial shapes and arrangement of prokaryotic
structure can be observed. The bright-field light microscope is an instrument that
magnifies images using two lens system. To observe the bacteria clearly, simple staining
will occur. In this experiment, four bacteria were stained and observed; Bacillus
megaterium, Saccharomyces, Escherichia coli, and Staphylococcus aureus.
Introduction
Due to bacteria’s lack of color and size, smear preparation and simple staining is
necessary. This is necessary because it allows the bacterial morphology to be observed. A
dried preparation of bacterial cells on a glass slide is a bacterial smear. Once a bacterial
smear is done correctly, the bacteria are evenly spread out on the slide yet separated from
one another, the bacteria are not washed o the slide during staining and the bacterial
form is not distorted. Then the bacteria can heat-fixed and killed in this way without serious
distortion of cell structure. A single stain or dye called a chromogen is used to create a
contrast between the bacteria and the background is referred to as simple staining. In this
experiment the dye that is used was methylene blue to create the contrast. The methylene
blue dye bind to bacterial cells by ionic interactions. The dye contains positively charged
groups and bind to negatively charged molecules like any proteins and the surfaces of
prokaryotic cells.
Procedure
1. Disinfect work area before and after you start the experiment.
2. Sterilize the inoculating loop and put a drop of water (distilled) on the slide using the
inoculating loop.
3. Sterilize the loop again and collect a small sample of yeast, making sure to barely touch
the agar disc.
4. Smear the yeast in the water that was placed on the slide. Make sure that it is a thin
smear and not milky. Let it air dry.
5. Burn the loop again.
6. Heat the slide 4-5 times until the yeast sticks to the slide (heat fix).
