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Posted: April 14th, 2023
Sepsis is usually caused by a bacterial infection; it can also be caused by fungal, viral or parasitic infections. Sepsis can cause blood clotting and inflammation. Blood clotting and inflammation in the body has life threatening consequences such as organ failure this may lead to death. Aseptic surgery was introduced in 1867 by Joseph Lister; the mortality rate of post operative surgery was reduced to 45%. The spray carbolic acid as biocide/germicide was used; biocide is a chemical agent that kills pathogens. The spray was used to dress and clean the wounds; in 1878 Robert Koch introduced the sterilisation of surgical equipment and dressing using steam.
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A bacterium is a prokaryotic microorganism that is found everywhere, the size of bacteria is usually a few micrometres in length they exist as millions. The shape of bacteria varies from rod, spiral, spherical. Hans Christian Gram in 1884 discovered that certain stains are taken up and retained by the cell. Eventually most of the bacteria were divided to two groups, gram-positive and gram-negative. The bacterial species was classified using the Gram stain test.
The Gram stain that emerge as blue or violet it is Gram positive, a gram-positive bacteria has a thick cell wall as there are many layers of peptidoglycan and teichoic acids. This means that gram stain can enter the cell and remain within the cell. If the Gram stain emerges as red or pink it is Gram negative, a gram negative bacteria has a thin cell wall that consists some layers of peptidoglycan, bounded by a second lipid membrane containing lipoprotein and lipopolysaccharides.
Escherichia coli (E. Coli) is facultative anaerobic gram-negative bacteria, E. Coli is rod shaped. The digestive system is affected by E. Coli where it causes bloody diarrhoea and abdominal pain. Staphylococcus aureus (S. Aureus) is sphere shaped gram-positive bacteria, S. Aureus can be found at several sites in humans such as nasal passages, skin, gastrointestinal tract and oral cavity. S. Aureus causes impetigo, mastitis and scalded skin syndrome. Antibiotic is a drug that inhibits the growth of bacteria or kill the bacteria. Antibiotics are from one class of antimicrobials and are harmless to the host hence used to treat infections. Antibiotics aren’t successful in treating fungal, viral and other nonbacterial infections. It is effective is treating bacteria but individual antibiotic varies on the type of bacteria. Microorganisms can become resistant towards antibiotics, hence withstand the effect of antibiotics, this is due to chromosomal changes or due to the exchange of genetic material via plasmids. This has been stimulated by the overuse of antibiotics for minor ailments (common colds) and unfinished course of antibiotics. There are many other antimicrobials such as antiseptic and disinfectants, Antiseptics are used to destroy or reduce microorganisms on the skin without damaging tissues; the common form of antiseptic is surgical rub. Disinfectants are used to disinfect inanimate objects such as surgical instruments. Disinfectant tend to have higher killing power as the chemicals used in disinfectants is for objects rather than the skin.
In order to research microbial growth, two experiments have been carried out. Experiment one was carried out to measure the effect of disinfectants and antiseptics on microbial growth. A mixture of antiseptics and disinfectants will be taken and the clearance zone will be measured on bacteria S. Aureus and E. Coli. Experiment two was carried out to screen the sensitivity of the multidisc system, this was done by placing two types of discs containing several antibiotics onto two types of bacteria E. Coli and S. Aureus.
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The mechanism of antimicrobial action of antiseptics and disinfectants has been researched by A. D. Russel from the Welsh school of pharmacy, the conclusion drawn is the target sites and concentrations of the antiseptic/disinfectant were the dependent factors.
The activity, action and the resistance of antiseptics and disinfectants has been studies by Gerald McDonnel and A. Denver Russel. The findings were that the antiseptics and disinfectants vary considerably even when the biocide levels are similar in each product. It has been found that there is some biocide resistance and cross-resistance with antibiotics.
A pre-prepared culture of E. Coli and S. Aureus grown in a nutrient broth is provided. Using each culture an inoculum was prepared by aseptically diluting 0.1ml of the each culture to a 9ml fresh nutrient broth. The culture was shaken to encourage mixing.
The spread plates were prepared by spread plating 0.1 ml of the inoculum on the surface of the nutrient agar. Two agar plates were prepared for each bacterium, four agar plates in total.
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The bottom of each plate was labelled with the name of the organism (i.e. E. Coli)
The eight antiseptic and disinfectant that was going to be tested in the experiment was numbered 1-8.
The bottom of the first agar plate for one of the bacterium was separated into four different sectors; a marker pen was used to label each sector 1-4. The bottom of the second agar plate was separated into four different sectors and labelled 5-8.
Repeat number 5 for the other bacterium agar plates.
The numbers on the bottom of agar plates link to the numbers on the antiseptic and disinfectant bottles.
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The tip of the forceps was sterilised by passing it through the Bunsen burner two to three times.
Using the sterile forceps the sterile paper disk was picked up and dipped into the disinfectant/ antiseptic that was numbered one.
It was made sure that the surplus disinfectant/ antiseptic had drained off before placing the disc into the agar plate. The disc was placed in the centre of sector 1of the S. Aureus inoculated plate
Another disc was placed onto the centre of sector one using the same disinfectant for the E. Coli inoculated plate.
Step number 8-11 was repeated for the remaining antiseptic/disinfectant. It was vital that the tip of the forceps was washed with alcohol and passed through the Bunsen burner to prevent cross mixing the disinfectant/antiseptic between each step.
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This is important as we were comparing the effectiveness of the disinfectant/antiseptic of the different types of organisms.
The sterile tip of the forceps was used to press the discs gently in order to ensure the disc has contact with the nutrient agar.
When the discs were placed into the four sectors of both agar plates, parafilm was used to seal.
The plates were inverted and incubated at 37°C for 24 hours.
After a week the plates were recovered and the diameter of the zones of the inhibition of the bacterial growth was measured in millimetres.
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As mentioned in the method of experiment one on how to produce spread plates for S. Aureus and E. Coli. This method was used to prepare another set of spread plates. (steps 1-2)
The bottom of each plate was labelled with the name of the organism (i.e. E. Coli)
The tip of the forceps was passed through the flame of the Bunsen burner two to three times in order to sterilise the tip of the forceps.
The multidiscs provided are of two types, multidisc M43 which is gram positive, M14 which is gram negative.
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The multidisc labelled M43 was placed in the agar plate labelled S. Aureus, gently press down on the disc using the forceps. The tips of the forceps are flamed in the Bunsen burner two to three times.
The multidisc labelled M14 was placed in the agar plate labelled E. Coli and gently press down on the discs using the forceps to make sure the discs are in contact with the agar.
The lids on agar plates were placed and sealed using parafilm. The plates were inverted and incubated at 37°C for 24 hours.
After a week the plates were recovered and the diameter of the zones of the inhibition of the bacterial growth was measured in millimetres.
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Experiment one studied the effect of Antiseptics and Disinfectants against the bacteria E. Coli and S. Aureus.
The most effective antiseptic used is Savlon as it has the largest zone of Inhibition for both bacteria’s, in the bacteria E. Coli the zone of inhibition is 18mm (table 1) and in S. Aureus the zone of inhibition is 25mm (table 1). The active compound is Savlon is Cetrimide which is a Quaternary Ammonium Compound (QAC), QAC is a surface active agent that has a hydrocarbon this is hydrophobic and there is another group which is hydrophilic. Surface acting agents are categorized into four areas depending upon the charge and ionisation of the hydrophilic group. The cationic agents show QAC’s; these agents are used as antiseptics and disinfectants as they have antimicrobial properties.
The least effective antiseptic is TCP as it has the least amount of zone of inhibition for both bacteria’s, for E. Coli the clearance is 20mm (table 1) and for S. Aureus the clearance is 14mm (table 1). The active ingredient in TCP is a Halophenol, Chloroxylenol is the main Halophenol used in antiseptic formulations as it’s bactericidal. The mechanism of action has not been studied in detail but as it is a phenolic character it is expected to be an antimicrobial
The most effective disinfectant towards the bacterium E. Coli is Wilkinson’s pine disinfectant as the zone of inhibition is 21mm (table 2), for the bacterium S. Aureus Dettol surface cleaner is the most effective disinfectant as the zone of inhibition is 37mm (table 2). Overall the most effective disinfectant is Dettol surface cleaner as the inhibition zone is S. Aureus is 37mm (table 2), the inhibition zone for E. Coli is 19mm (table 2). The active ingredient in Dettol surface cleaner is Chloroxylenol which also is a Halophenol.
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The least effective disinfectant was Zoflora spray as the inhibition zone in the bacterium S. Aureus is 26mm (table 2) and in E. Coli it is 17mm (table 2). The active ingredient in Zoflora spray is Benzalkonium chloride which is a QAC therefore a cationic surface acting agent.
In addition, several compounds contain QAC’s such as Mr muscle and Wilkinsons pine Disinfectant but the zone of inhibition varies this may be due to additives present. The additives would dilute the concentration of QAC present hence decreasing the zone of inhibition.
Experiment two studied the sensitivity of the multidisc system with the bacteria S. Aureus and E. Coli.
The multidisc M43 contains antibiotics that are sensitive to S. Aureus bacteria. The most effective antibiotic towards the gram positive S.Aureus bacteria is Penicillin G as the zone of inhibition is 28mm. Penicillin G works by inhibiting the formation of the bacterial cell wall, this is done by blocking the cross-linking of cell wall structure.
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The least effective antibiotic in the M43 disc is Trimethoprim and Sulphamethoxazole, the zone of inhibition is 0 mm. Trimethoprim and Sulphamethoxazole work by inhibiting enzymes that is needed in the production of Folic acid, which is an essential compound in the cell. Therefore, the antibiotics can kill bacteria but the process will be very slow. If we incubated the agar plates for longer zone of inhibition may be seen. However, S. Aureus may have become resistant to these antibiotics. If there is partial resistance to either of the antibiotics, the bacteria can be killed by the combination of these two antibiotics.
The multidisc M14 contains the antibiotics that are sensitive to E. Coli bacteria. The most effective antibiotic towards the gram negative E. Coli bacteria is Cotrimoxazole as the zone of inhibition is 28 mm. Cotrimoxazole is a combination of Trimethoprim and Sulphamethoxazole which works by inhibiting enzymes producing Folic acid, which is an essential compound in the cell.
The least effective antibiotic towards E. Coli is Ampicillin as the clearance zone is 0 mm (Table 4) Ampicillin works by inhibiting the formation of cell wall by blocking the cross linking of cell wall structure. The antibiotic is resistant towards E. Coli due to chromosomal changes or due to the exchange of genetic material via plasmids. As S. Aureus is a gram positive bacterium, in gram positive the cell wall structure is made of peptidoglycan as well as polysaccharides or teichoic acids. The antimicrobial only has to cross one membrane hence the zone of inhibitions is higher in the S. Aureus plates for both experiments one and two.
Overall, antimicrobials effectiveness is affected by many factors, some of which are temperature of incubation, concentration of the antibacterial, molecular weight influences crossing the membranes, thickness of membrane and by which mechanism the antibiotic kills or inhibits the bacterial growth.
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In conclusion, this experiment would have to be repeated several times in order to test reproducibility, and then a clear conclusion could be made. Many antimicrobials are potentially toxic therefore advice should be sought if unsure. Also, as we can see that disinfectants and antiseptics can vary considerably in spite of similar level of active ingredient. This calls for a closer inspection of claims made by companies regarding their products.
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