Antibiotic Dosage Effects on Bacteria

John HeagyPittsburgh Central Catholic

Grade 11

Ampicillin

• Antibiotic derived from the penicillin group to kill bacteria

• Treats many different types of infections caused by bacteria, such as ear infections, pneumonia, and E. coli or salmonella infection

Escherichia coli

• Rod-shaped, 2 micrometers diameter

• Gram negative bacteria• Survival, growth, and replication

require only a single carbon source and ammonium salts.

• Most common prokaryote model.

Yeast

• Easy to grow and culture• Unicellular• Most studied cell in the world• Saccharomyces cerevisiae• Similar cell cycle, biochemistry and genetics to other

eukaryotic cells, like those in humans

Staphylococcus Epidermidis

• Gram positive coccus.• Common surface symbiont in many mammals

(including humans).• Most forms considered non-pathogenic.• Potentially pathogenic upon systemic entry.• Forms biofilms.

Streptococcus

• Streptococci are Gram-positive, non-motile cocci that divide in one plane, producing chains of cells

• The streptococci are catalase negative (unlike Staphylococcus) and may be either facultative or obligate anaerobes

• Streptococci are the bacterial infection that causes strep throat

Gram Negative Gram Positive• Cell wall is thin extra

layer of lipopolysaccharide which adds extra level of protection.

• This outer membrane protects the bacteria from several antibiotics.

• Simple cell wall.

• Antibiotics such as penicillin work against the formation of the cell wall.

Purpose To determine the effects of ampicillin on different kinds of bacteria.

Null Hypothesis Hypothesis

The dosage of ampicillin will have no significant effect on the growth of the bacteria.

The dosage of ampicillin will have a significant effect the growth of the bacteria.

Materials• 80 Plates• Yeast• Staph• Streptococci• E. Coli• Ampicillin stock• Test Tubes• Sterile pipette tips and Micropipettes• Vortex• Sidearm flask• Spreader bar• Ethanol• Micro burner• Test tubes• Test Tube Rack• SDF Test Tubes• Micro tubes• Incubator

Procedure

• 1) Bacteria (E. coli, Staph, and Streptococci) was grown overnight in sterile LB media.

• 2) Yeast was grown overnight in sterile YEPD media.• 3) A sample of the overnight culture was added to fresh

media in a sterile sidearm flask.• 4) The culture was placed in an incubator (37°C) until a

density of 50 Klett spectrophotometer units was reached. This represents a cell density of approximately 10^8 cells/mL.

• 5) The culture was diluted in sterile dilution fluid to a concentration of approximately 10ˆ5 cells/ml.

Procedure(Cont.)• 6) Ampicillin was mixed with the appropriate amount Sterile

Distillation Fluid to create ampicillin concentrations of 1x, .1x, .01x, and 0x, x being the 20mg/mL concentration of ampicillin.

• 7) The solutions were vortexed and allowed to sit at room temperature for 15 minutes.

• 8) Each of the above concentrations were pipetted onto 5 replicates of plates.

• 9) The culture was placed in an incubator (37°C) for 24 hours to allow for growth.

• 10) The resulting colonies were counted visually. • 11) The appropriate statistical analyses were performed to

adequately assess the hypothesis.

Chart of Test Tube Components

Control .01x .1x x

Microbial 0.1 ml 0.1 ml 0.1 ml 0.1 ml

Sterile Dilution Fluid (SDF) 9.9 ml 9.807 ml 9.807 ml 9.87 ml

Ampicillin 0 ul 3 ul (2mg/ml) 3 ul 30ul

Total Volume 10 ml 10 ml 10 ml 10 ml

ANOVA

Streptococcus Staph Yeast E. Coli

P-Value 0.030935 1.67E-11 1.06E-10 1.29E-11

Significance Significant Significant Significant Significant

Stock Concentration vs Number of Colonies

Control 1% stock 10% stock 100% stock0

20

40

60

80

100

120

140

160

180

200

Streptococcus

E. Coli

Yeast

StaphNum

ber o

f Col

onie

s

Stock Concentration

P= 0.030935

P= 1.06E-10

P= 1.29E-11

P= 1.67E-11

Dunnett’s TestT-value T-crit Significance

Staph 3.29

x 17.63 Significant

.1x 5.02 Significant

.01x 0.88 InsignificantStreptococcus

x 2.98 Insignificant

.1x 1.61 Insignificant

.01x 0.236 InsignificantYeast

x 15.57 Significant

.1x 4.78 Significant

.01x 0.62 InsignificantE. Coli

x 16.41 Significant

.1x 3.49 Significant

.01x 1.03 Insignificant

Conclusion

• The ANOVA results appeared that the variance between and within the groups was significant, but the Dunnett’s tests appeared to have mixed results.

• The streptococcus culture results were insignificant for every concentration, which could be a result of a resistance to antibiotics.

• The regular dosage and .1x dosage of ampicillin was significant in all other microbials.

• The .01x concentration appeared to have no significant effect on the microbial growth.

Limitations Further Testing

• Plating was not exactly synchronized, which could possibly result in varying colony counts.

More microbial could be tested.

Test dosages higher than x to see if there is a greater detrimental effect on growth.

Find an LD-50

Sources

• "E. coli." Kids Health from Nemours. Web. 28 Oct. 2009. <http://kidshealth.org/kid/stay_healthy/food/ecoli.html#>.

• <http://web.uconn.edu/mcbstaff/graf/Student%20presentations/S%20epidermidis/sepidermidis.html>.

• http://www.rxlist.com/principen-drug.htm• http://www.cehs.siu.edu/fix/medmicro/strep.htm• Woese, C. R. (1987). "Bacterial evolution". Microbiological

reviews 51 (2): 221–271. PMC 373105. PMID 2439888