1. Effect of Media and pH
on the Gram Stain
Results of Bacteria Over
Time
By: Gabrielle Bonner
Research Mentors: Dr. Bolyard and Cathy Huggins
B

2. Introduction
Gram NegativeGram Positive Heat Fixation
Grams crystal
violet
Lugol’s iodine
Decolorization
(ethanol)
Safranin
• What is Gram staining?

3. Introduction
• Differences in Gram stain results are due to
content of the cell wall
http://www.microbiologybytes.com/introduction/graphics/Prokaryotes4.gif

4. Introduction
• Unknown protocol at Union University Microbiology Lab
• Bacterial cultures are recommended to be Gram stained between
12-18 hours of age
• The peptidoglycan layer of the cell of some Gram positive
bacteria gets thinner (Beveridge 1990)
• Gram negative bacteria can resist decolorization (Gregersen 1978)
• Gram variable- mixture of both Gram positive and negative cells
http://labmedx.ucsfmedicalcenter.org/labmanual/clinlab/micro_images/ba
cteriology/bacteria%20A-H/Bacillus-sp-cap-gs-check-5_s.jpg

5. Introduction
• Is this 12-18 hour time frame
absolute/appropriate?
• Are there environmental factors that may change
the Gram stain results of bacteria?
• Jeffery Hirtes (2013) - observed the effect of
temperature on Gram stain results

6. Research Objective
The objective of this
experiment was to test the
effects of media and pH on the
Gram stain results of bacteria
over time.

7. Methods
• 7 bacteria were tested
• 4 Gram positive
• Bacillus cereus
• Sarcina aurantiaca
• Kocuria rhizophilia
• Bacillus megaterium
• 3 Gram negative
• Escherichia coli
• Acinetobacter calcoaceticus
• Neisseria flava
http://www.buddycom.com/bacteria/gnr/acinetc1259.jpg
http://atlas.microumftgm.ro/bacteriologie/bactsp/neisseria/frotiu/pi
c/ng_m_cp_3.jpg

8. Methods
• Aseptic Technique

9. Methods
• Three different types of media
• nutrient agar
• tryptic soy
• circle grow

10. Methods
• 3 pH’s: 6,7,8
• The pH of each media type was adjusted to 6 and 8

11. Methods
• Nine media tubes for each bacteria

12. Methods
• Acinetobacter calcoaceticus-worked with under a hood

13. Each of the 7 Bacteria
Circle growNutrient agar Tryptic soy
Methods
6 7 8pH:
Media:
6 7 8 6 7 8
• Slides were made at 16,18,20, 22, and 24 hours of culture age
• Repeated experiment

14. Methods
• Gram stains were performed on the bacterial slides in each
condition

15. Methods
• Binocular compound microscope
• Oil immersion

16. Results
• Media or pH did not change the Gram stain results
• Bacillus cereus, Kocuria rhizophilia and Sarcina aurantiaca
remained Gram positive
• Acinetobacter calcoaceticus and Escherichia coli remained
Gram negative

17. Results
• Bacillus megaterium stained Gram variable
• Mixture of purple and pink cells at each media, pH and time
combination

18. Results
• Neisseria flava stained Gram variable
• Stain was the same with each combination of media, pH and time
http://www.idimages.org/images/organisms/A00023/A00023_01_m_lab.jpg

19. Results
• Acinetobacter calcoaceticus
• Shape varied according to the growth media
• Did not grow in circle grow media
Coccus shaped in nutrient agar Bacillus shaped in tryptic soy
agar

20. Results
Bacteria Bergey’s
Manual Gram
stain
classification
Gram stain
observed
Did pH change
the Gram
stain?
Did media
change the
Gram stain?
Did the Gram
stain change
over the 16-24
hour time
period?
E.coli negative negative no no no
A.
calcoaceticus
negative negative no no no
Neisseria flava negative variable no no no
Bacillus cereus positive positive no no no
Kocuria
rhizophilia
positive positive no no no
Sarcina
aurantiaca
positive positive no no no
Bacillus
megaterium
positive variable no no no

21. Discussion
• Bacillus megaterium stained Gram variable within the 12-18
recommended age
• (Beveridge 1990)- Bacillus brevis stains positive in the start
phases of growth and stains variable/negative in the exponential
and stationary phase

22. Discussion
• Neisseria flava
• Classified as Gram negative according to Bergey’s Manual
• Cells can resist decolorization
• Brodie et al. (1971)- Neisseria can be encapsulated
• Mrs. Huggins did not include it in unknown project

23. Discussion
• Acinetobacter
• Bergey’s Manual does classify it as a coccobacillus
• Rod shaped in the lag and exponential phases of growth
• Spherical shaped in the stationary phase of growth
• Laffeineur et al. (2002) – claims that Acinetobacter resists
decolorization and can look purple

24. Discussion
• Interestingly, B. cereus did not stain Gram variable at any point
• Observations suggest that can be a wider window of time in
which Gram stains can be performed

25. Future Research
• Observe Actinomyces and Arthrobacter (Beveridge 1990)
• Determine the conditions under which N. flava can resist
decolorization
• Acinetobacter and other bacteria can be studied to determine
the conditions in which they change shape
• Determine ratio of purple and pink cells in B. megaterium over
time

26. Acknowledgements
• Lord and Savior Jesus Christ
• Dr. Bolyard
• Mrs. Huggins
• Dr. Wofford
• Dr. Kerfoot
• Dr. Thierfelder and Dr. Madison
• Family and Friends

27. Literature Cited
Bergey’s Manual of Systematic Bacteriology. 1984.
Beveridge, T. J. 1990. Mechanism of Gram variability in select bacteria. Journal of
Bacteriology 172:1609-1620.
Brodie, E. J.L. Adler, A. Daly. 2002. Bacterial Endocarditis Due to an Unusual Species of
Encapsulated Neisseria: Neisseria mucosa endocarditis. The American Journal of
Diseases of Children 122(5): 433-437.
Gregersen, T. 1978. Rapid method for distinction of Gram negative from Gram positive
Bacteria. European Journal of Applied Microbiology and Biotechnology 5:123-127.
Laffineur, Kim, Michele Janssens, Jacqueline Charlier, Veranique Avensani, Georges
Wauters, and Michel Delmee. 2002. Biochemical and susceptibility tests useful for
identification of nonfermenting Gram-negative rods. Journal of Clinical Microbiology
40:1085-1087.

28. Questions?