Crystal violet solution (10 ml of gentian violet, saturated alcohol solution and 40 ml of ammonium oxalate 1. 0% aqueous solution); Gram’s iodine solution (1 g of iodine crystals, 2 g of potassium iodide, and 300 ml of distilled deionized water); safranin solution, saturated aqueous solution; Bunsen burner; slide and cover slip; deionized or distilled water; 95% alcohol and; Bibulous paper. For the second part of the experiment were preparations of tetracycline concentrations(0. 1 µg L-1, 1.
0 µg L-1, 10 µg L-1). II. Procedure At the commencement of the experiment, each bench received four culture tubes that contained a mixture of Staphylococcus and Escherichia bacteria. These two cultures were immediately mixed and distributed to ensure that all experimental set ups had similar bacterial compositions. With four tubes apportioned each experimental group, a single test tube was used in Gram stain to determine the relative proportions of Staphylococcus and Escherichia in the mixed culture.
Part I: Staining Techniques When bacteria is stained by the Gram Stain procedure, they exhibit two salient and distinct characteristics: Gram positive bacteria such as Staphylococcus hold the crystal violet stain in their cell walls making them appear blue while the Gram negative bacteria such as Escherichia coli are decolorized and show a red counterstain after they are rinsed in an alcohol solvent; usually safranin.
This technique of bacterial species differentiation is most valuable and accurate when dealing with fresh cultures that are still in their active replication phase but it loses considerable sensitivity as cultures age making them barely dependable (Alcamo 1997). In old cultures, biochemical and serological identification techniques are done after Gram stains to increase the accuracy of species identification. Procedurally, the culture was swirled to suspend the bacteria then a pipette was used to draw a small volume of water.
A single drop was placed at the center of a clean and sterilized glass slide and the culture spread evenly to cover an area that is approximately ? inch squared. This was then allowed to air dry and consequently fixed by repeated passing of the slide over a burner flame. Care should be taken to avoid heating up the slide and consequently causing denaturization or at worst burning the bacterial culture. After this, the slide was flooded with crystal violet and held for roughly thirty seconds to one minute before rinsing the slide with deionized water.
The sample was then covered with Gram’s iodine and held again for thirty seconds to one minute before rinsing with deionized or distilled water. The next stage involved sample decolorization in 95% alcohol for 20-30 seconds it was rinsed in deionized water. Counter staining was done with safranin. For an effective counter staining process it is prudent to allocate an approximate reaction time of ten seconds. The ensuing result was again rinsed with distilled water to wash off excess stain pigments on the slide then dried with bibulous paper. For identification of the Gram stain result, the slide was observed under the microscope.
Observably, Gram positive organisms stained purple black while Gram negative organisms stained red or pink. In line with the nature of the mixed culture, Staphylococcus stained purple black hence Gram positive and Escherichia stained pink hence Gram negative. Part II: Culturation with Different Concentrations of Tetracycline. Taking the original four tubes per bench, the first tube was seeded with 0. 1 µg L-1, the second; 1. 0 µg L-1, and the third; 10 µg L-1. The fourth tube was left without tetracycline seeding so as to act as a control for the experimental set up.
All these tubes were returned to the tube rank situated on the side bench. After the seeded cultures had settled they were placed into the incubator and the temperature for incubation set at 37? C. On the report board, the proportions of all laboratory pairs of Staphylococcus and Escherichia. However, given the understanding that the proportion of Escherichia is simply one proportion of Staphylococcus, the final result presentation only included results of Staphylococcus proportion with respect to the varying concentrations of tetracycline. These final results were tabulated as follows;
Results In comparison to the control tube, Staphylococcus proportions in the three tetracycline seeded test tubes showed a quantitative decrease from bench one to bench eight and with increasing tetracycline concentrations. In Bench 1 where the control showed a proportion of 85, this proportion decreased to 65 in the 0. 1 µg L-1 tube; 15 in the 1. 0 µg L-1 tube; and 10 in the 10 µg L-1 tube. In bench 2 where the control tube recorded a proportion of 80, this proportion decreased to 55 in the 0. 1 µg L-1 tube; 20 in the 1. 0 µg L-1 tube; and 5 in the 10 µg L-1 tube.
In bench 3 with a control proportion of 90: the second highest value in all the benches, there was a decrease in proportion to 60 in the 0. 1 µg L-1 tube; 25 in the 1. 0 µg L-1 tube; and 5 in the 10 µg L-1 tube. In bench 4 with a control proportion of 75 there was a decrease in proportion to 70 in the 0. 1 µg L-1 tube; 25 in the 1. 0 µg L-1 tube; and 10 in the 10 µg L-1 tube. Tube 5 results were as follows; control; 95 (the highest in all the benches), 65 in the 0. 1 µg L-1 tube; 15 in the 1. 0 µg L-1 tube; and 15 in the 10 µg L-1 tube. Tube 6: control; 80, 50 in the 0. 1 µg L-1 tube; 20 in the 1.
0 µg L-1 tube; and 5 in the 10 µg L-1 tube. Tube seven: control; 90, 50 in the 0. 1 µg L-1 tube; 25 in the 1. 0 µg L-1 tube; and10 in the 10 µg L-1 tube and lastly tube 8: control; 80, 65 in the 0. 1 µg L-1 tube; 25 in the 1. 0 µg L-1 tube; and 5 in the 10 µg L-1 tube. Since initially all the cultures subdivided in the benches originated from a single original culture inoculated in the same media and subjected to similar experimental influences, calculating the mean bacterial proportion the eight benches yields; Average control Staphylococcus proportion; 84. 375. Staphylococcus proportion in 0.
1 µg L-1 tube; 60. 0, in the 1. 0 µg L-1 tube; 21. 25 and in the 10 µg L-1 tube; 8. 75. In line with these results there is an observable decrease in Staphylococcus proportions with increasing tetracycline concentrations. Tetracycline is a broad spectrum antibiotic that is effective against a wide range of Gram positive and Gram negative bacteria. Tetracyclines are bacteriostatic agents that inhibit bacterial protein synthesis by binding irreversibly to the 30S subunit of the bacterial ribosomes. They inhibit the binding of the enzyme aminoacyl tRNA to the ribosomal acceptor site on the mRNA ribosome complex.
Through this action, the addition of amino acids onto the growing polypeptide chain is stalled. Tetracycline passively diffuses through the outer membrane before being actively transported into the cytoplasm by energy dependent mechanisms (Yaffe & Aranda 2004). Due to the wide use of these antibiotics they are frequently released into the environment where they may stimulate co-resistance or cross resistance between genetically related microorganisms on one hand and resistance to a host of other antibiotics and even heavy metals.
Resistance occurs due to an increase in the concentration of tetracycline in the cell. With the aid of the tet gene, bacteria can develop resistance to tetracycline by increasing the cells capacity to efflux the antibiotic out of the cell wall. Additionally the alteration of the tetracycline attachment sites can also be used as a resistance mechanism. For this reason environmentally isolated microorganisms do possess a significant level of resistance making them tolerant to concentrations that are lethal to purified cultures.
In the research the increase of antibiotic susceptibility of Staphylococcus from extremely low tetracycline concentrations to significantly higher concentrations directly correlates with the decrease in Staphylococcus proportions. This observation implies that as tetracycline concentrations increase, it inhibitory effect on the target sites also increases up to a point where, autolysis and subsequent apoptosis occurs. However, this is only possible if levels of concentration used in the study are way above the levels normal therapeutic dosage.
In such a case gradual inhibitory mechanisms may clear the whole of bacterial composition. Alternatively, environmentally isolated Staphylococcus can be able to withstand even effective therapeutic dosages in vitro due to higher antibiotic resistance levels. In the latter case, lethal concentrations are extremely high and sometimes unhealthy for human or animal prescriptions. On the basis of the results therefore, an exponential decrease in tetracycline dosage dilution to the next level may reduce the proportion of Staphylococcus to insignificant levels.
Moreover, the effects of tetracycline observed only correlate with the differences in the initial susceptibility of Staphylococcus to tetracycline which obviously differed from bench to bench. Assuming that the considerable susceptible dosing increased for some appreciable duration, the accumulation of the drug may have stimulated the tetracycline resistance mechanisms. Other studies, such as Sanders et al (1976), have shown that should the dosing be discontinued then the effects on bacterial composition may persist long after the discontinuation of the dosing.
Therefore, despite the paucity of data on the efficacy of long term acting tetracyclines against Methicillin Resistant Staphylococcus aureas (MRSA) preliminary findings have demonstrated that long acting tetracyclines may be the best pharmacotherapeutic option for individuals suffering from some types of MRSA infections.
References
Alcamo, I. Edward. (1997). Schaum’s Outline of Theory and Problems of Microbiology McGraw-Hill Professional, p. 37-42 Chopra, I. & Howe, T. B. G. (1978). Bacterial Resistance to Tetracyclines.
Microbiological Reviews, Dec. 1978, Vol. 42, No. 4, p. 707-724. American Society of Microbiology Patenaude, L. Erica. , Atoyan, A. Janet, Potts, A. David, & Amador, A. Jose. (2008). Effects of Tetracycline on Water Quality, Soil and Gases in Aerated and Un-Aerated Leachfield Mesocosms. Journal of Environmental Science and Health Part A. 43, 1-10. Taylor & Francis Group, LLC. Panniker, & Ananthanarayan (2004). Textbook of Microbiology. 11th Edition. Blackswan. p. 193-194 Roy, H. Frederick. , Fraunfelder, W. Frederick.
, & Fraunfelder, T. Frederick. (2007). Roy and Fraunfelder’s current ocular therapy. Edition 6. Elsevier Health Sciences. P. 28-29 Sanders, C. C. , Sanders, W. E. , Harrowe, J. D. (1976). Bacterial interference: effects of oral antibiotics on the normal throat flora and its ability to interfere with group A streptococci. Infect Immun. 1976 March; 13(3): 808-812. Yaffe, J. Summer & Aranda, V. Jacob. (2004). Neonatal and Pediatric Pharmacology: Therapeutic Principles in Practice. Lippincott Williams & Wilkins, p. 417-420