Basics of Microbiology Lab: Gram-Staining
Lesson 8 of 12
Objective: Students will be able to perform a Gram Staining Assay in order to determine if a contamination has occurred in a bacterial culture presumed to be sterile.
Engage (Activate Student Thinking)
SAY: "Quality Control believes that something is awry in our yogurt production facility!"
ASK: What specific characteristics or conditions would cause you to suspect that contamination has occurred in the yogurt section of our dairy processing plant? (Possible student responses: We could observe a pungent odor, color change, or odd taste)
Explore (Guided/Student-Centered Activity)
ASK: What are our standard operating procedures when we suspect a potential contamination has occurred? What evidence can we use to confirm or deny a contamination? (Possible student responses: We must contain and isolate the suspected contaminated yogurt culture and perform a gram-stain to determine presence of a pathogen or microbe.)
1. Students should view the BioRad Gram Staining training video posted online.
2. While viewing the video, create a flowgram of the standard operating procedures conducted when performing a Gram Stain Assay. Record the illustrations and captions for each drawing of the flowgram on the template provided.
Explain (Formulate Ideas)
ASK: How could we discover the origin of a possible contamination in the yogurt section of the dairy processing plant?
Virtual Gram Staining Lab - http://virtuallab.nmsu.edu/stain.php
Objective: In order to perfect our gram staining laboratory technique and use those skills to address the issue of possible contamination in our dairy processing plant, students will practice with an initial, virtual lab. Go to http://virtuallab.nmsu.edu/ and select the “Gram Staining” lab. Address the following inquiries:
1. What harmful bacteria can end up in yogurt if not handled correctly?
2. Describe what good and bad yogurt look like.
3. Why are air bubbles a sign of contamination?
4. What specific purpose was Gram staining developed for? What is it used for today?
5. What color are Gram-negative bacteria? Gram-positive?
6. Why do bacteria stain pink or purple?
7. What is phosphate buffer solution?
8. What is crystal violet?
9. What is Gram’s iodine?
10. What is the purpose of the 95% alcohol solution?
11. Why is it important to have the alcohol sit for no longer than 30 seconds?
12. What is Safranin?
Elaborate (Apply and Extend Understanding)
As we come to the end of this pre-lab component of this experience, this is the portion of the lesson where students get to show off how much they have learned. By preparing the following T-Tables students are able to demonstrate proficiency in the use of this laboratory technique BEFORE completing the actual hands-on assay. This progression of activities has been a great saver of time and resources in addition to accelerating students mastery of core laboratory techniques.
I. Write detailed procedures for how to prepare your slide for staining in your laboratory notebook using a T-Table or Cornell Notetaking strategy. Students should have no less than 10 steps. (This T-table should be used during the actual, hands-on Gram Staining Assay.)
II. Write the detailed staining procedures in your laboratory notebook using a T-Table or Cornell Notetaking strategy. Students should have no less than 8 steps in each student-generated procedure.
Evaluate (Monitor Understanding)
In order to further assess student proficiency of this technique students can both view a Gram Stain Animation and complete an assessment on laboratory applications in Microbiology. Students are required to answer all five questions in the short quiz correctly in order to gain access to the community lab and I monitor the scores attained by circulating and offering guidance and assistance when needed. Based on the availability of necessary resources and materials, skill level of students, as well as time constraints, instructors have the option of conducting an actual gram stain assay with students and then proceed to a post-lab reflection OR proceed directly to the post-lab reflection after completion of the virtual lab.
A. Basic Yogurt and Bean Simple Staining Lab (Hands-on Lab/Practicum)
B. Advanced Gram Stain Lab (Hands-on/Practicum)
- 45 blank microscope slides and cover slips (3 slides/group)
- Oil immersion microscope (preferable) Light microscope w/400X will do
- 15 dropper bottles of 95% ethanol
- 15 test tube holders or clothespins
- 90 60x15mm Petri dishes
- 1 box of latex gloves
- 15 Bunsen burners
- Box of Kim wipes
- Ethanol/acetone rinse
- Immersion oil
- Biohazard bags
- Parafilm and scissors (one set for the class)
- 1 each per lab group: Gram’s Crystal Violet, Gram’s Iodine, and Gram’s Safranin stains
- 1 each per lab group: 24 hr. agar and broth cultures of Alcaligenes faecalis, Bacillus cereus and Serratia marcescens
NOTE: It is important to use bacterial cultures that are between 24-48 hours old. If they are too old, they may all appear Gram (-). However, if they are too young, they may show a mixture on Gram (+) and Gram (-) when it is actually a pure culture.
1. Describe the non-contaminated sample.
2. Describe the contaminated sample.
Below are all common mistakes made during gram staining. For each mistake write what you would expect to see or have seen.
- Slide not heat-fixed:
- Slide over heat-fixed:
- Slide over-decolorized:
- Slide under-decolorized:
- Smear too thick:
- Insufficient time for Safranin counterstain: