Experiment 6, 10, 11: Ubiquity of Bacteria, Smear Preparation, and Simple Staining
January 20, 2026
Emily Adams
Objectives
Ex. 6: The objective is to culture bacteria from natural environmental sources and
recognize that both the types and abundance of bacteria differ depending on the environment
sampled. This exercise also emphasizes the concept that bacteria are ubiquitous and present in
nearly all environments.
Ex. 10: The objective is to learn how to properly prepare a thin bacterial smear on a
microscope slide using cells from both liquid and solid media. Students will also practice
heat-fixing the smear to preserve cell structure and ensure that the bacteria adhere to the slide for
staining.
Ex. 11: Students should be able to prepare and stain a bacterial smear using a simple
stain. This exercise focuses on understanding why staining is necessary for visualizing bacteria
under a brightfield microscope and on observing basic bacterial morphologies such as shape and
arrangement.
Relevant Techniques/Reagents
Techniques: In this laboratory, techniques for culturing and visualizing bacteria will be
performed. These include collecting and culturing bacteria from natural environmental sources,
preparing thin bacterial smears from both liquid and solid cultures, heat-fixing bacterial cells to
microscope slides, and applying a simple staining technique. Microscopic observation using a
brightfield microscope will be used to examine bacterial cell morphology/arrangement.
Reagents/Media: The reagents and media used in this lab will include nutrient agar plates,
a TSA plate, a blood agar plate, liquid and solid bacterial cultures, a simple stain such as
methylene blue, and distilled water.
Organisms
Staphylococcus epidermidis, is a Gram-positive bacteria and is considered the most
resistant of all non–sprore-forming pathogens (p. 576).1
Corynebacterium xerosis, is a Gram-positive bacteria that normally lives in the eye, skin,
and mucous membranes (p. 623).1
Bacillus megaterium, is a Gram‑positive, primarily aerobic bacterium commonly found in
soil and diverse environments.2
Basic Procedural Outline
Ex. 6: Obtain and label a nutrient broth tube with your initials and an object you wish to
investigate. Moisten a sterile swab by immersing it into a tube of nutrient broth and getting rid of
excess broth by pushing the swab against the inner wall of the tube. Rub the moistened swab
over some object and return the swab to the tube of broth. Next for the TSA plate, label the plate
with initials and your chosen environmental choice. For the blood agar plate, label the bottom of
the plate with initials and the body part chosen to investigate. Rub a water-moistened swab over
an external body part. Then, incubate the plates (inverted) and tube at 37°C for 48 hours.
Ex. 10: Broth Cultures. Obtain a clean slide and make a target circle on the bottom of the
slide. Shake the culture vigorously and transfer two loopfuls of organisms to the center of the
slide and spread over the area of the target circle and allow it to dry by evaporation. Then place
the slide in a clothespin and pass several times through the Bensen burner flame.
Ex. 10: Slants and Plates. Obtain a clean slide and make a target circle on the bottom of
the slide. Use a cooled inoculating needle (was previously flamed), to pick up a small amount of
organisms and mix it into water on the slide. Flame inoculating needle before placing back in
holder. Allow the mixture on the slide to dry by evaporation. Then place the slide in a clothespin
and pass several times through the Bensen burner flame.
Ex. 11: Prepare heat-fixed smears of S. epidermidis, B. megaterium, and C. xerosis. Then
stain the bacterial smear with methylene blue for 1 minute, then briefly wash the stain off with
water, and then have the water drops blotted off with bibulous paper. Examine the slides with
oil-immersion and compare with photos in figures 11.4 and 11.5. Record observations.
References
1. Talaro, K. P, & Chess, B. (2021). Foundations in Microbiology (11th ed.). McGraw-Hill.
2. Cappa, M., Perego, C., Terzis, D., & Principi, P. (2025). Bacillus megaterium favours CO₂
mineralization into CaCO₃ over the ureolytic pathway. Scientific Reports, 15(1), Article
21861. [Link]