CONTROL OF BACTERIAL GROWTH WITH CHEMICALS AND DISINFECTANTS


AIM 

The aim of this experiment was to determine the way the different chemical agents affect microbial growth and evaluate some common antiseptics and disinfectants`s effectiveness in either reduction or inhibition of the bacterial growth. 

HYPOTHESIS 

It is expected that in the end of this experiment no growth is observed in the samples treated with zephiran or alcohol. Additionally for the second experiment no growth is expected to develop in the parts where the finger is treated by detergent or detergent and alcohol, while in the parts where the finger is not treated with detergent, microbial growth is expected.  

INTRODUCTION 

Bacterial growth is the scientific term used to describe the increase of the number of the bacterial cells and the time that this process takes to realize is called the generation time, which is determined during the exponential phase. The bacterial growth is mainly affected by the the physical factors that include pH, osmotic pressure, concentration of oxygen, temperature and moisture level and the nutritional factors that include the concentration of the elements in the microorganisms`s habitats such as: carbon, nitrogen, phosphorus, sulfur, vitamins and microelements [1]. 

Antimicrobial agents are important agents about the control of the bacterial growth and there are different types according to the objects they are applied to, spcecifically: disinfectants applied to nonliving objects and antiseptics applied to living ones [2]. 

Sterilization is the process of eliminating each microorganism in a certain object leading to the condition of sterility. The number of the microorganisms present is proportional with the time needed to eliminate all of them. Additionally, disinfection is the process of reducing the number of the microorganisms present that can show a possible risk degree. The agents used for disinfections are the disinfectants that exhibit those properties: effectivenes to reduce the number of microorganisms, inexpensive, not having an unpleasant odor, able to penetrate the microorganisms and fast to perform the process of disinfectation [3]. 

The effectiveness of an antimicrobial agent is measured by the degree of effectivity, which is affected by: time, pH, temperature and concentration. Temperature and concentration of the antimicrobial agent are directly proportional to the effectiveness of the antimicrobial agent. The high concentrations of the antimicrobial agent act as bactericidal that kill the microorganisms, while the low concentrations act as bacteriostatic that only inhibit the growth of the microorganisms [4]. 

Chemical antimicrobial agents are able to denaturate proteins of the microorganisms and the denaturation can exist in two different forms: bacteriostatic or bacteriocidal. The bacteriostatic denaturation changes the structure of the proteins, but this change is recoverable; while the bacteriocidal denaturation cause a non-recoverable change in the structure of the proteins, therefore leading to the death of the microorganisms. Another group of chemical antimicrobial agents include the surfacants, which are soluble compounds that perform the reduction of the the surface tension, that can disrupt the different proteins or lipids present in the plasma membrane of the microorganisms. This group includes detergents, alcohols and quaternary ammonium solutions [5].  

Phenol Coefficient is the standard used about comparative studies needed to determine the effectiveness of any disinfectant, where a coefficient greater than 1 defines an agent with effectiveness lower than that of phenol and vice versa, while a coeficcient of 1 expresses the same degree of effectiveness like that of phenol [6]. 

There are different types of chemical antimicrobial detergents and they differ from each other, from the material they are applied to and their functioning mechanism. Soaps are able to eliminate the microorganisms due to their consistency on alkali and sodium. Detergents that are of two types according to their charge, either positive (cationic) or negative (anionic) eliminate the dirt and microbes when used together with water. One of the mostly used cationic detergents was Zephiran (benzalkonium chloride) that is an quaternary ammonium compound, whose effectiveness is decreased because of the soaps, calciums or magnesium ions [7].  

Other methods used in disinfection are the halogens, heavy metals, alcohols, phenols that denature proteins of the microorganisms and destroy their enzymes, oxidizing agents that disrupt disulfide bonds in proteins and their membrane structures, alkylating agents that disrupt the structure of nucleic acids and proteins, dyes that interfere with the replication of DNA of the microorganisms and different plant oils [8].  

MATERIALS & METHODS 

Materials 

  • Pippettes and tips 
  • Forceps 
  • Pins 
  • Bunsen Burner 
  • 1% Zephiran 
  • LB broth containing tubes 
  • Overnight 2% E.coli culture 
  • 70% Ethanol 

Method 

Experiment1 

Each of the groups performed the same experiment but different exposure times in the presence of the detergent. Each of the groups is provided with 2 different tubes, one consisting of distilled water and one consisting of overnight 2% E.coli culture and 4 pins inside it. Initially the pins inside the tube are taken off from the tube by using a forceps. Later the pins are dried by the help of a filter paper and positioned in different sterilants, initially in 1% zephiran for 30 minutes (our group`s time) and later 1 minute in distilled water. Afterwards, the pins were positioned in a nutrient broth and placed overnight in heater under the temperature of 37oC. 

Experiment2 

Initially a Petri dish`s upper plate was divided in 4 equal zones and labelled with 4 different letters for each part, reciprocally: A, B, C and D. In each zone a different action is performed related to the experiment. In A, unwashed finger is pressed over the agar of this zone. In B, washed, but undried thumb is pressed. In C, washed and dried thumb is pressed, while in D, thumb sterilized with 70% Ethanol and dried on the air is pressed over this zone`s agar. Later this Petri dish is incubated overnight under the temperature of 37oC. 

RESULTS 

Table1: The results of Use-Dilution Method 

Disinfectants  Zephiran  Ethanol  No disinfectants 
Time (min)  10  20  30  10  20  30  Control sample 
Group 1  No growth            +++ 
Group 2    No growth          +++ 
Group 3      No growth        +++ 
Group 4        No growth      +++ 
Group 5          No growth    +++ 
Group 6            No growth  +++ 

 

DISCUSSION  

The results of the samples treated by use-dilution method (Table 1) are compatible with the results expected when hypothesis was defined. Therefore the hypothesis should be accepted and this leads to the conclusion that both zephiran and ethanol are satisfactory methods to prevent the microbial growth. The reason why ethanol is successful in killing microorganisms, respectivally E.coli in this experiment, is because of its ability to denaturate proteins and dissolve the lipid membrane of the bacteria [9].  

The reason why zephiran is successful in killing microorganisms, respectivally E.coli in this experiment, is because of its ability to damage the cytoplasmic membrane of the microorganisms, proteins and DNA as well [10].  

In this experiment, control samples`s growth was controlled as well. The difference in that experiment was that they were treated with sterile water and not with any type of disinfectants (Table 1). Their growth was normal, as no growth inhibitor factor was induced there.  

In the second part of the experiment, the results of the experiment (Fig.1 & Fig.2) are not compatible with the results expected according to our hypothesis. In the figure 1  of the result part, it is seen that in the A part no bacterial growth has occurred although bacterial growth was ecpected there due to the hypothesis and the fact that unwashed finger contains various of microorganisms. In the B part growth has occurred, although no or very little growth was excpected there, as the finger applied in the broth was washed with detergent. In the C part,  very little growth has occurred and this result is not compatible with what was expected as well. In the D part very little growth has occurred, although no growth was expected. The results of this part of the experiment are contradictory with the hypothesis and the scientific literature related to the way applicaion of detergents, drying the washed hands and sterilization with alcohol reduces or inhibits the microbial growth [11]. The reason why this has occurred can be either the detergent and the alcohol efficiency is really low or in an error in the labelling the parts related to the process that would be performed.  

In the result of the other experiment related to the evaluation of the microbial growth in 4 different parameters using alcohol and handwash (figure 2), it is seen in the A part little growth has occurred. In B part very little growth has occurred. In the C part, very little growth has occurred, while in the D part 1-2 microorganisms`s growth can be seen. This experiment seems more compatible to the result expected in the hypothesis as when the handwash detergent is applied in the B part, the growth of bacteria has reduced. Additionally the growth has reduced in a satisfactory level 1-2 microorganisms when the finger is disinfected with alcohol.  

REFERENCES 

  1. http://www.sas.upenn.edu/LabManuals/biol275/Table_of_Contents_files/9-Antimicrobials.pdf retrieved 30.04.2016 from Internet source 
  1. “Bacteria.” Microsoft Encarta. 2001 edition. CD-ROM. Microsoft Corporation, 1993-2000 
  1. Rodowski, Jr., Christopher A. “Disinfectant” The World Book Encyclopedia. 1995 vol. 5 (D) 
  1. http://www.utm.utoronto.ca/~w3bio152/webpage_fall/lab_files/lab_02_bacteria.pdf retrieved 30.04.2016 from Internet Source 
  1. American Society for Microbiology “Bacteria” October 18, 2002
    http://www.microbe.org/microbes/bacterium1.asp retrieved 30.04.2016 from Internet Source 
  1. http://highered.mheducation.com/sites/0072320419/student_view0/chapter7/study_outline.html retrieved 01.05.2016 from Internet Source. 
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  1. http://generalbacteriology.weebly.com/sterilization-and-disinfection.html retrieved 03.05.2016 from Internet source 
  1. http://scienceline.ucsb.edu/getkey.php?key=2160 retrieved 03.05.2016 from Internet source 
  1. http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit2/control/antibio.html retrieved 03.05.2016 from Internet source 
  1. http://www.cdc.gov/features/handwashing/ retrieved 03.05.2016 from Internet source 

 

 

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