Exercise 7: Negative Stain
Introduction
Certain bacteria are unable to maintain their natural shape and arrangement after heating. The negative stain is used to view cellular morphology without the influence of heat-fixing. The specimen is mixed into a suspension in a negative stain. An acidic dye is used as the chromogen. The negatively charged, acidic chromogen is repelled by the cell’s proteins and other structures leaving the cell unstained against a dark background. One of the most common acidic dyes used for a negative stain is nigrosin that provides a purple background. It may be used to stain delicate cells such as spirochetes so that their shape and size can be properly measured.
Course Intended Outcomes
Perform and understand the basis of different types of microbiological staining techniques (e.g., simple stain and gram stain) to characterize and identify bacteria.
Materials Used
Students will work in small groups. Each group will need the following materials for this exercise.
- Bunsen burner
- Inoculation loop
- Test tube rack
- Bottle of nigrosin stain
Additional materials and cultures will be assigned by your instructor
Procedure
Technique:
- Apply 1 drop of nigrosin stain toward one end of a clean slide.
- Use a sterile inoculating loop to add a small amount of culture to the drop of nigrosin. Emulsify the culture into the stain but do not spread out the suspension.
- Using the short edge of a second clean slide press it against the slide at a 40 degree angle. Next, draw it backward into the suspension.
- When the suspension spreads along the edge quickly push the suspension across the surface of the bottom slide.
- Allow the bottom slide to dry completely then view with the microscope.
- Disinfect the top/spreader slide with bleach then wash with detergent and return to your slide box.
- Do NOT heat fix.
Sampling/Inoculation:
- Your instructor will assign the cultures to be used for inoculation by your group.
- Collect the required materials for the lab.
- Label your slides using a wax pencil with the organism initials (e.g., EC = Escherichia coli). This will prevent mixing up your slides and help with slide orientation.
Lab Clean-up
Wash slides with detergent, rinse and dry. Return slides to your slide box and store in the bin labeled for your section.
Return bacteria cultures to the front bench.
Thoroughly clean your microscope and return it to the lab prep room.
Disinfect your benchtop and wash your hands before leaving the lab.
Results and Interpretation
Record your results in your lab notebook creating a table as shown.
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Specimen Name / Source |
Background Color |
Cell Color |
Shape |
Arrangement |
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Critical Thinking Questions
Why are the cells spread in a suspension instead of applying the stain to an air-dried smear?
What is the reason that the cells remain colorless after applying the stain?
The image illustrates the preparation of a bacterial smear on a microscope slide in five stages, arranged from left to right across the top row and then continuing along the bottom. First, a small blue drop of liquid is placed near one end of a clean glass slide. Next, a small sample of bacterial culture is transferred into the drop and mixed.
A second slide or spreading tool is then positioned at an angle against the drop. The angled slide is drawn across the surface to spread the liquid into a thin, elongated smear. The final stage shows the completed blue smear distributed evenly across the center of the microscope slide, ready for drying, fixation, and staining.
