The Effect of Strong Agents on Gram Positive and Gram Negative Bacteria

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Table of Contents

Abstract
Materials
Procedure
Staining
Results
Discussion
Limitations and sources of errors
Improvements
References

Abstract

This experiment is set to investigate the effect of strong agents such as tetracycline on the composition of bacterial community. The relative proportions of bacteria are examined after being subjected to tetracycline of varying concentrations.

The process was undertaken by first of all staining compositions of bacteria with staphylococcus and Escherichia.The cultures were then subjected to known quantities of tetracycline and incubated at room temperatures. There was a notable reduction in abundance of gram positive bacteria under large quantities of tetracycline as compared to the smaller quantities. This has an implication that tetracycline has little effect on gram positive bacteria. According to the Canadian journal of microbiology, tetracycline has the ability to interfere with the process of protein synthesis in bacterial cells. This can therefore result in death of bacteria hence a decline in their abundance.

Materials

The procedure required cultured bacteria (staphylococcus and Escherichia) which were stained using crystal violet and iodine. Safranin was used as a counter stain in the procedure after discoloration with ethanol. Three different concentrations of tetracycline (0.1, 1.0, and 10 µg L^-1) alongside a zero concentration were also used in the procedure. The microscope and slides were used to perform the counts while a pipette was used to draw some water.

Procedure

All the benches received four culture tubes containing a mixture of Staphylococcus and Escherichia bacteria. These cultures were mixed immediately before lab, such that laboratory cultures had similar bacterial composition. Each laboratory pair took one of the four tubes. Each laboratory pair performed a Gram stain using their culture tube to determine the relative proportion of these bacteria at the beginning of the experiment (time 0). Each bench also coordinated a Gram stain and counting of the fourth control tube.

Staining

The culture was swirled to suspend bacteria. This was followed by drawing of a small volume of water using a pipette. A small drop of the culture was then placed on a slide. Moisture was eliminated through air drying. The slide was quickly passed through a flame for fixing the cells. The cells were then flooded with crystal violet for one minute. A drip of water was then used until the purple streams ran clear. Iodine solution was added to the slide for three minutes after which water was dripped on the cells for one minute.Decolourization then followed using ethanol with also a drip of water for one minute.

A counter stain, Safranin was then applied to the cells for 1-2 minutes followed by a drip of water till when the slide was clear. The slides were then observed under the microscope. The observations made were recorded in table 1 Each pair received a known quantity of tetracycline to their culture. At each bench, pair 1 added 0.1 µg L^-1, pair 2 added 1.0 µg L^-1, and pair 3 added 10 µg L^-1.. No amount of tetracycline was added to the fourth control slide. The cultures were finally well labeled and incubated at optimal temperatures of 37degrees for two weeks.

After the two weeks another gram stain was performed on the cultures. Relative proportion of Staphylococcus (Gram positive) and Escherichia (Gram negative) in the culture was estimated. The data on the proportion of Staphylococcus was recorded in table 2.

Results

After 2 weeks period it was observed and recorded that the relative abundance of gram positive staphylococcus bacteria had reduced. The decline in abundance of bacteria corresponded to the concentrations of tetracycline in the cultures. For instance in bench one there was notable decline from 90 to 60 -15- 10 corresponding to 0 , 0.1ugL, 1.0ugL and 10.0 ugL concentrations of tetracycline respectively.

Week 2 results.

Week 1 initial	control	0.1ugL	1.0ugL	10.0ugL
Bench 1	90	60	15	10
Bench 2	90	55	20	5
Bench 3	95	60	25	5
Bench 4	95	70	25	10
Bench 5	90	65	15	15
Bench 6	95	50	20	5
Bench 7	85	55	25	10
Bench 8	85	65	25	10

Discussion

From the results obtained after the 2 weeks period it is clear that indeed tetracycline had an effect on gram positive bacteria. In the control experiment there was no difference after a 2 weeks period because of lack of tetracycline in the bacteria culture. The effect of tetracycline on gram positive bacteria was evident in the decline in numbers or abundance after the 2 weeks period. The significant decline in abundance of gram positive bacteria at 1.0ugL and 10ugL is indicative of the fact that strong agents like tetracycline have an effect on the abundance of the bacteria.

This is so because tetracycline interferes with the structure of ribosomes in the bacteria hence interfering with the process of protein synthesis in the cells of bacteria. Bacteria are killed in this process as a consequent of lack of proteins in the cells. This results in the decline in their abundance at these high concentrations of tetracycline. The high sensitivity of the gram positive bacteria towards tetracycline is a confirmation that indeed tetracycline is still an effective antibiotic in treating bacterial infections.

The results of this experiment are indeed in tandem with already published materials concerning the treatment of some bacterial infections using tetracycline as an antibiotic. Tetracycline is a broad spectrum antibiotic but studies have identified some oral species of bacteria have shown resistance to the antibiotics. This experiment has demonstrated that the bacteria used were susceptible to tetracycline and therefore agrees with the already established facts. (Gladwin et al 2007)

Limitations and sources of errors

There were shortcomings with regard to handling of apparatus and reagents that might have led to alteration of expected results.The observation of the number of bacteria under the microscope was characterized by estimation of close figures that represented the abundance of the gram positive bacteria. During the fixing of bacterial cells on the slide by sliding it over fire there is a possibility that some bacteria were burnt and thus contributing to the alteration of the anticipated results.

During the counterstaining process with Safranin , a difference in the time between two and three minutes would make some cells to remain unstained and therefore could not be observed after the two weeks growth period. During the suspension of the culture if care was not taken properly there is a possibility that the adequate number of bacteria was not maximized before staining the slides. The main source of error in the procedure concerns the various steps in staining the bacteria before incubating for two weeks.

Overstraining was likely to interfere with the observed number of bacteria. On the other hand under-staining was likely to leave other bacteria out of the count and hence altering the results of the experiment.

Improvements

The observations under the microscope should be undertaken using a number of magnifications to arrive at the appropriate figure that can provide reliable results on the proportions of gram positive bacteria. Care has to be taken during fixing of the cells, the slide has to be passed over the flame without burning some of the cells on the slide.

Suspension of the bacteria should be done carefully by adequately swirling the culture to obtain a mixture with the maximum abundance of bacteria. Counterstaining should be performed at a predetermined duration of time to avoid varying results of the experiment due to some cells not getting the counter stain. A more elaborate procedure can be set up to investigate specific strains of staphylococcus that have developed resistance to tetracycline. With well performed staining procedure the effect of tetracycline can be examined on the specific strains of gram positive bacteria. This can be a major break though and can provide consumers with reliable information on the usefulness of tetracycline antibiotics with treatments of specific diseases and infections. (Gladwin et al 2007)

References

Canadian Journal of Microbiology.

Gladwin, Mark; Bill Trattler (2007). Clinical Microbiology made ridiculously simple. Miami, FL: MedMaster, Inc. pp. 4–5.

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Livermore DM. Bacterial resistance: origins, epidemiology, and impact. Infect Dis 2003;36 (Suppl 1):S11-23.

Lipsitch M, Levin BR. The population dynamics of antimicrobial chemotherapy. Antimicrob Agents Chemother 1997;41:363-73.

Madigan, MT; Martinko J, Parker J (2004). Brock Biology of Microorganisms (10th Edition ed.). Lippincott Williams & Wilkins.

Gentilini E., Denamiel G., Llarente P., Godaly S., Rebuelto M., DeGregorio O.: Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. J. Dairy Sci., 2000, 83, 1224-1227.

Roberts, M. C., and G. E. Kenny. 1986. Dissemination of the tetM tetracycline resistance determinant to Ureaplasma urealyticum. Antimicrob. Agents Chemother. 29:350-352.

Khan, S. A., and R. P. Novick. 1983. Complete nucleotide sequence of pT181, a tetracycline resistance plasmid from Staphylococcus aureus. Plasmid 10:753-765.

Watts J.L., Salmon J.A.: Activity of selected antimicrobial agents against strains of Staphylococcus aureus isolated from bovine intramammary infections that produce β-lactamase. J. Dairy Sci., 1997, 80, 788 – 791.

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