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In 1884 Hans Christian Gram, a Danish bacteriologist, attempted to find a universal stain that would work with all bacteria. In the process, he discovered that bacteria could be divided into two different groups -- one that retained a stain, called 'gram-positive,' and one that didn't, called 'gram-negative.' His unique method for identifying these two groups became the first step in any bacterial identification process. Even the simple determination that a bacteria specimen is gram-positive or gram- negative can direct a doctor in diagnosis, as different bacteria cause different diseases. For example, the bacteria that causes scarlet fever is gram-positive, while that which causes typhoid or cholera is gram-negative.
Gram staining helps doctors make a diagnosis, but can it also help suggest a cure? What is the relationship between gram classification and antibiotic use? Do common antibiotics interact differently with gram-positive and gram-negative bacteria? Answer these questions through experimentation.
Question: Will four common antibiotics (Penicillin, Ampicillin, Neomycin, and Erythromycin) have the same effect on both gram-negative and gram-positive bacteria?
Observe/Gather Data: Do some research to find information about antibiotics and gram staining, so that you can make an informed hypothesis.
Hypothesis: Based on your research, write a detailed hypothesis predicting the answer to the question.
Experiment: An experiment to test your hypothesis will need two parts. In part one, perform a gram stain on bacteria cultures to determine which are gram-negative and which are gram-positive. In part two, set up a controlled experiment to measure the effect of each type of antibiotic on each type of bacteria. Working with chemicals and bacteria can be hazardous. Before you start, read the following safety note.
Often the chemicals used to prepare slides may be toxic, corrosive or have other related hazards. Always carefully read the entire label before using a chemical. Be sure you understand the hazards involved, the proper safety equipment to wear, and what you will do in case of a spill or contact with your skin. The stains used for the gram stain process will discolor clothing and skin. Basic safety equipment that you should wear include safety goggles (splash type), chemically resistant gloves, and a chemically resistant lab apron. Work in a clean, well ventilated, uncluttered area where you can quickly wipe up spills. Always keep chemical bottles tightly capped.
While most environmental bacteria are not harmful to healthy individuals, once concentrated in colonies, they can be hazardous. To minimize risk, wear disposable gloves while handling bacteria, and thoroughly wash your hands before and after. Never eat or drink during bacteria studies, nor inhale or ingest growing cultures. Work in a draft-free room and reduce airflow as much as possible. Keep petri dishes with cultured mediums closed—preferably taped shut—unless sampling or disinfecting. Even then, remove the petri dish only enough to insert your implement or cover medium with bleach or 70% isopropyl alcohol. When finished experimenting, seal dishes in a plastic bag and dispose. Cover accidental breaks or spills with bleach or alcohol for 10 minutes, then carefully sweep up, seal in a plastic bag, and discard.
Gram stain kit (contains crystal violet stain, Gram iodine stain, ethyl alcohol solvent, Safranin O counterstain, plain microscope slides, medicine dropper, coverslips)
Some of the steps of the gram stain process are hard to carry out perfectly. To practice, it is a good idea to make a 'control' slide. Try collecting some bacteria from between your teeth (using a toothpick) and placing it on a slide with a drop of water. If the Gram staining procedure is done correctly, your slide should have a mixture of gram-negative and gram-positive cells as well as some neutrophils (white blood cells) with pink nuclei. After you have tried that, stain each of your live bacteria cultures using the following procedure:
Once you have determined which of your live cultures is gram-negative and which is gram-positive, label them clearly and move on to the next part of the experiment.
One way to test bacteria susceptibility to antibiotics is to use the Kirby-Bauer or 'disc diffusion' method. This method involves measuring the inhibition of bacteria growth around an antibiotic disc placed in a culture.
Analyze Data/ Form Conclusions
Analyze your data. How did each antibiotic perform in each bacteria culture? Were the antibiotics more effective overall against gram-positive or gram-negative bacteria? What were the limitations of your study? Could you get more accurate results if you tested a larger number of bacteria cultures?
Form conclusions. Did your results support your hypothesis? Why or why not? What do your results tell you about the process of prescribing antibiotics? How could you continue this study to discover even more about the relationship of antibiotics and bacteria?
Don't work with antibiotic discs if you are allergic to those forms of antibiotics.
The bacteria you work with can also be hazardous. Always wash your hands thoroughly before and after handling the bacteria cultures. Washing them before will minimize contamination of the bacteria cultures you are growing. Washing them afterwards will minimize your exposure to harmful bacteria that may be growing in your cultures.
When you have finished studying a culture, pour enough household bleach into it to cover the bottom of the dish. Then cover the culture, seal it in a plastic bag, and throw it away.