Lab: Identifying Bacteria by

Gram Staining
Lab Design created by Katie Rowley
Objective:
You will determine the shape and cell wall makeup of
six different bacteria by conducting a Gram stain
technique and viewing slides using a compound
microscope (1000X total magnification oil immersion
objective).
Question:
Which of the unknown bacteria plates (A,
B, C, D, E, F) is Escherichia coli, Bacillus
cereus, Micrococcus luteus, Serratia
marcescens, Rhodospirillim rubrum,
Branhamella catarrhalis?
Research the Problem:
1. Name and draw the three (3) common shapes of bacteria.
(Include their scientific root name.)

2. What is the size range for most bacteria?

3. Differentiate between Gram positive and Gram negative

bacteria.
 4. Research information on the following bacteria:
Gram +/Gram
-/Stained
Where are Color? Cell wall thick
Bacteria they found? Shape Size or thin? Other Info?

E. coli

B. cereus

M. luteus

B. catarrhalis

S. marcescens

R. rubrum
Procedures:
DAY ONE: HOW TO PREPARE UNKNOWN BACTERIA SLIDES FOR GRAM STAINING:
1. Obtain 6 clean microscope slides.

2. On the frosty part of the slide, write the following information using a permanent pen:

* Unknown bacteria plate letter (A, B, C, D, E, or F)

* Group name or initials

* Class period

* Date

3. Place transparent tape on top of the label, but do not wrap it around the slide.
4. With a permanent pen draw a circle in the middle and the bottom of the slide.

5. With a plastic pipet add one drop of Phosphate Buffer Saline Solution (PBS) inside the circle
on top of the slide.

6. Obtain a sterile flat toothpick and pick up one colony or a small amount of bacteria from
unknown plate or test tube labelled “Unknown Bacteria A”. Gently stir and smear the small
amount of bacteria in the drop of PBS in the circle on the top of the slide.
7. Place slide on the slide attachment of bacterial sterilizer
machine. CAUTION: HOT! Do not touch machine! Teacher
will show you how to use device safely.
8. Let prepared bacterial circle heat dry. This technique will
kill the bacteria and “fix” it in the circle of your slide. NOTE:
You must fix your bacteria to the slide in order to conduct a
Gram stain.
9. Repeat steps 2-8 for the remaining unknown bacteria
samples (plates/test tubes B-F).
DAY TWO: HOW TO GRAM STAIN FIXED BACTERIA SLIDES

1. Add about 5 drops of Hucker’s Crystal Violet to the culture. Let

stand for one minute. Bacteria will stain purple. Wash briefly with
water and shake off excess.
2. Add about 5 drops of iodine solution to the culture. Let stand for 30
seconds, wash briefly with water and shake off excess.
3. Tilt slide and decolorize with solvent (acetone-alcohol solution) until
purple color stops running. Be careful not to over-decolorize. Wash
immediately (within 5 seconds) with water and shake off excess
4. Add about 5 drops of Safranin O. Let stand for one minute, wash
briefly with water and shake off excess.
DAY THREE: HOW TO VIEW BACTERIA USING OIL IMMERSION
METHOD WITH A COMPOUND MICROSCOPE

https://www.youtube.com/watch?v=f7KlFSgdUGU

Instructions: Pay close attention to this video on how to use the 100X objective oil
immersion to view bacteria on your prepared Gram stained slides.

Note: You can damage the other objective lenses by getting oil on them. If you
have any questions, please ask.
Observations: Draw and color what you see on each slide
and paste into notebook.

A B C

D E F
Data Table 1: Draw and fill-out data table in notebook.

Color after Gram positive Name of

Unknown plate Shape of Gram or Gram unknown
Bacteria staining? negative? Bacteria

F
Write a Conclusion:

● Purpose
● Make a claim(s)/Answer the scientific question.
● Support claim(s) with evidence. Include the shape and
Gram results to support answers.
● Provide scientific explanations.
● Write two possible errors.
● Write two possible procedural improvements. Hint:
Reliability? Validity?
● State one practical application of results.