- Take one bottle of the samples of bacteria you want to inoculate e.g. Ecoli and using the 2ml syringe withdraw about 1ml of the bacteria. Note that before you withdraw the bacteria from the bottle, flame the neck of the bottle using the Bunsen burner, this is an aseptic technique.
- Using your syringe with the 1ml of Ecoli, add it to one of t the petridish containing the agar and immediately place back the lid.
- To sterilise your glass spreader and forceps, put a little bit of alcohol in a beaker and place them in this alcohol.
- Remove your glass spreader after some few minutes from the alcohol and pass it through a flame from the Bunsen burner.
- Use the sterilised glass spreader to spread the inoculated Ecoli in the petridish.
- After this, pass the glass spreader again through the flame before placing it back in the beaker with the alcohol.
- Take out the forceps in the alcohol and as before with the glass spreader, pass it through flame to sterilise.
- Use the sterilised forceps to pick the antibiotic disk.
- Lift the lid of the inoculated petridish with Ecoli and place the antibiotic disk in the middle of the dish using the forceps.
- Pass the forceps through flame again before placing it back into the alcohol.
- Using the sticky tape seal the dish by taping the lid and the dish together.
- After sealing the dish, incubate it for one week between 25 to 30 degrees centigrade.
- Repeat all this steps for each of the remaining 2 bacteria i.e. bacillus and micrococcus.
(xv) Finally when the results are ready, place the dish on a piece of graph paper and use the squares to measure the diameter of the clear area.
(d)Changes I had made in my method from my plan:
- The amount of bacteria that I inoculated in my method was 1ml as opposed to the 0.5ml I used in my planning I made this change because0.5ml is too little to have enough bacteria growth on the agar.
- I incubated the sealed dish for one week ( 7days), instead of 2 to 3 days so as to give enough time for the antibiotics to see more clearly on how they will affect the growth of the bacteria.
(e) Health and safety procedures:
- The syringe that you are going to use to inoculate the bacteria should not have any needles. This is because needles can penetrate your skin and if any pathogenic organisms penetrate your body, it might cause harmful diseases or death.
- Use disinfectant like sudol to disinfect your working area. You do this by spraying the table with disinfectant and with a tissue dry the table.
- Any scratched or cracked glass wear like boiling or test tubes should be disposed. This is because this might lead to you cutting yourself and and therefore microorganisms penetrating your body.
- Alcohol is highly flammable and therefore use it in small quantities and away from the naked flame.
- Never bring your hand into contact with your mouth when dealing with microorganisms this is because your hands are contaminated and might lead to microorganisms penetrating your body. Wash your hand with disinfectant before leaving the laboratory.
(f) Results:
(i) Ecoli.
(ii) Bacillus subtilis.
(iii) Micrococcus luteus.
(g) Review and evaluation of my experiment and result:
- The results of my first experiment was inconclusive because there was no growth of bacteria on most part of the dish even the places without antibiotics. This was mainly because the bacteria samples we were given was not enough. But as shown in my result table, I repeated the experiment 4 times.
- From the result we can clearly see that not all of the antibiotics had effect on the growth of bacteria. The only antibiotic that heard effect on the growth of all the 3 bacteria was Gentamicin (GM).
- The Bacteria that was most affected by the antibiotics was Ecoli where 5 antibiotics stopped the growth of antibiotic.
- From my research I found out that PG that is penicillin had a very great effect on Bacillus and from my result this is not the case. This therefore means that there was something wrong most likely with the bacillus sample.
- N.B. I have noticed from my result that penicillin had no effect on bacillus that is Gram positive, and from my research I found out that penicillin are active primarily against Gram positive because Gram-negative bacteria are impermeable. This means that I had not inoculated enough bacillus sample in to the nutrient agar.
(h) Problems encountered doing the experiment:
- In my first experiment, the amount of bacteria samples we were given was very little. This made it difficult to retrieve the bacteria with the 2ml syringe. To over come this you use a larger volume of bacteria sample.
- The other problem occurred when I was heating the neck of the bottle containing the bacteria sample the bottle was a little too hot to handle.
(I) Discussion:
Microorganisms are organisms that can’t be seen with the naked eye and you need microscope to see them.
Bacteria: This is a type of microorganism that is found in a wide range of environmental condition. It is a single celled organism.
Bacteria can be
- Saprophytic – They feed on degrading organic material from the soil and water.
- Parasitic – They live on a host such as animal and plant.
- Photosynthetic – They synthesise food through light absorption and photosynthesis.
- Finally some bacteria gain their energy from inorganic chemical oxidation.
There are 2 main types of bacteria;
- Achaebacteria – This are possibly the earliest life forms and are thought to be very old e.g. methane generating bacteria.
- Eubacteria – These were developed later and include e.g. all gram-positive bacteria.
Before these 2 main groups were formed, bacteria were grouped according to shape and movement. These include.
- Ecoli – This was first isolated by Eschecheria in 1885 from the feces of an infant. It was found later to be a normal inhabitant of the intestinal tract of the human body. The longevity of Ecoli is reduced outside the animal body. Characteristics of Ecoli are
- They are short rods which can be motile or non motile.
- They are used for fermentation of glucose and lactose to produce acid and gases such as carbon dioxide and hydrogen.
- It is Gram positive.
- It is occasionally pathogenic to man and can cause diseases in the intestinal tract.
- They are aerobic.
- Bacillus subtilis – The members of the genus bacillus are universally distributed in soil and water. They are not formed in animal body. Characteristics of Bacillus are.
- Almost all the species are Gram positive.
- They are capable of forming heat resistant spores.
- All the members are saprophytic and usually not pathogenic with the exception of anthrax bacillus.
- The members are typically aerobic but some can grow in almost complete absence of oxygen.
- They are none motile.
- They cause Anthrax disease that is treated with penicillin.
- Micrococcus luteus – Their cells are spherical or ovoid occurring in short or long chains. They are found in the soil and some of the characteristics are,
- It is an obligate aerobic organism with typical respiratory metabolism.
- They have the ability to grow in media with high salt and this provides a simple means of isolation.
- It produces acid from glucose aerobically.
- They are known to synthesize hydrocarbons.
- Exposure to sunlight in the air is lethal to non-pigmented strains of micrococcus but not to yellow pigmented strains.
(j) Antibiotics:
These are chemical substances produced by certain type of microorganisms that are active against other microorganisms. They are natural product of microbial activity. Some antibiotics can be made more effectively by chemical modification; these are said to be synthetic. Gram-positive bacteria are usually more sensitive than Gram negative although some antibiotics act only on Gram-negative bacteria.
N.B. An antibiotic that acts on both Gram-negative and positive bacteria is called a broad-spectrum antibiotic while one that acts on a particular type of bacteria is called narrow spectrum antibiotic.
(k) Industrial production of antibiotics:
The mass production of antibiotics began during World War II with streptomycin and penicillin. Now most antibiotics are produced by staged fermentations in which strains of microorganisms producing high yields are grown under optimum conditions in nutrient media in fermentation tanks holding several thousand gallons. The mold is strained out of the fermentation broth, and then the antibiotic is removed from the broth by filtration, precipitation, and other separation methods. In some cases new antibiotics are laboratory synthesized, while many antibiotics are produced by chemically modifying natural substances; many such derivatives are more effective than the natural substances against infecting organisms or are better absorbed by the body, e.g., some semisynthetic penicillins are effective against bacteria resistant to the parent substance.
Production of Penicillin in Industrial Fermenters (Bioreactors)
Antibiotics such as penicillin are usually produced in large cylindrical vats, constructed of stainless steel, containing a liquid medium in which Penicillium chrysogenum is grown.
Withstands heat / allows heat to pass through
Before use, fermenters must be sterilised, usually with superheated steam.
Usually these fermenters are operated in a batch process.
After a certain amount of time for fungal growth, followed by gradual production of antibiotic, the contents are removed and processed to extract the antibiotics, then the fermenter is cleaned, sterilised and the process is repeated.
Penicillin extraction
After 6-8 days of batch culture, the liquid medium is pumped out, filtered and concentrated. The basic antibiotic - benzyl penicillin - is precipitated as crystals when potassium compounds are added.
This antibiotic may then be modified by the action of other micro-organisms or by chemical means, before being mixed with inert substances and pressed into tablets or converted into syrup or injectable form.
Although the molecular structure of penicillin is known, and it may be synthesised by chemical methods, it is not economic to do so. The production process still relies on fungal fermentation based on biological principles, although modern strains are much more productive than the early strains. This has been achieved through screening programmes involving isolates from different sources, and treatment to encourage mutations.
(l) Industrial application of antibiotics.
- Pharmaceutical product of microbial origins.
From an economical point of view the pharmaceutical industry is probably the most important industry in using microorganisms.
Microorganisms produce virtually all the medically important antibiotics and antibiotics are one of the major classes of pharmaceutical products. It is in the pharmaceutical industry that some of the most important application of the new gene technology has been made, although most of these advancements have not involved antibiotics.
Major products that are either currently manufactured or are anticipated to be manufactured via genetically emerged microbes are hormones such as insulin and human growth hormone.
N.B. Because of the high potential of economic value of pharmaceutical agent it can be anticipated that most of the new advancements will be in this field.
(m) Conclusion on my results from both the table and the bar chart:
- From the result in both the graph and the table we can see that even though that not all the antibiotics had effect on the bacteria some had effect on the growth of the bacteria on the agar and. We can safely say that antibiotics are chemical substances produced by certain microorganisms that are active against other microorgarnisms.
- From the bar chart we can conclude that
- Four antibiotics have effect on the growth of Ecoli and they are; CD, GM, T, E and FC. FC was the highest active on Ecoli because it had the largest diameter of0.95mm and that means that FC had killed the largest amount of Ecoli around it.
- Two antibiotics had effect on the growth of bacillus on the agar and they are; GM and T. GM had the largest diameter of 1.65mm and therefore was the most effective on halting the growth of bacillus on the agar.
- Finally also in micrococcus only two antibiotics had effect on its growth in the agar and they were; PG and GM. PG had the largest diameter of 1.1mm and therefore that means PG had killed most of the bacteria around it.
Therefore from my result we can conclude that;
- GM and T are broad-spectrum antibiotics because they act on both Gram positive and negative bacteria.
- PG is a narrow spectrum antibiotic because it acts on a particular type of bacteria.
(n) Bibliography:
- www.yaho.com/edexcel/pencil.htm.
- www.synysccc.edu/academic/mst/microbes/09mlute.htm.
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