SENSITIVITY OF FLOOR MICROFLORA TOWARDS VARIOUS DISINFECTANTS

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Lalitha et al. SJIF Impact Factor 6.647 Volume 6, Issue 3, 1316-1328 Research Article ISSN 2278 4357 SENSITIVITY OF FLOOR MICROFLORA TOWARDS VARIOUS DISINFECTANTS Dr. Ch. Lalitha*, N. Yerusha, Ch. Mutyala Rao, S. Bhagya Sria and D. Roopa Devi *Lecturer in Microbiology, Dr.V.S.Krishna Govt. Degree College(A), Visakhapatnam. Students, Microbiology UG, Dr.V.S.Krishna Govt. Degree College(A), Visakhapatnam. Article Received on 20 Jan. 2017, Revised on 08 Feb. 2017, Accepted on 01 March 2017 DOI: 10.20959/wjpps20173-8914 *Corresponding Author Dr. Ch. Lalitha Lecturer in Microbiology, Dr. V.S. Krishna Govt. Degree College (A), Visakhapatnam. ABSTRACT Microorganisms are omnipresent. They may be useful (e.g. Lactobacilli) or harmful (e.g. Pseudomonas, Salmonella etc) for human beings. The warm and moist environment of our surroundings including floors provides optimal conditions for their growth. There are many microbial species commonly present on the floor surfaces (Serratia marcescens, Staphylococcus aureus etc), most of which are non-pathogenic or pathogenic at higher concentrations. In order to control growth of such organisms floor disinfectants like Lizol, Domex, Dettol are commonly used. According to the Indian Medical Association, Lizol and Dettol are claimed to be the most effective disinfectants. In practice, it is impossible to maintain the conditions of the floor as it is maintained in vitro. Therefore, in the present study, experiments were designed using in vivo analysis to confirm the findings. A survey was conducted to determine consumer s preference and preferred products were further analysed for their effectiveness in inhibiting the growth of floor microorganisms (in vivo). Attempts were also made to study the longevity of these floor disinfectants using turbidometric analysis. Our results suggested Phenyl to be the more effective as compared to other products used for the investigation. KEYWORDS: Disinfectant, Floor Microflora, in vitro, in vivo, Pathogens, Turbidometric method. INTRODUCTION Microorganisms, the friend and foe of human being are present inside and outside the human body. Moisture present on the human body serves to be a perfect medium for the growth of www.wjpps.com Vol 6, Issue 3, 2017. 1316

numerous organisms. It is necessary to maintain hygienic conditions for effective reduction in the potential infection caused by such organisms. [1] Hygiene is defined as a science concerned with the prevention of illness and maintenance of health, whereas, personal hygiene is a condition promoting sanitary practices. To keep a check on the growth of microorganisms, routine hygiene practices using commercially available or medicated personal hygiene products are generally followed. [2] There are in numerous microorganisms growing on our skin surface and even after use of soap or an antiseptic, these cannot be fully eradicated. Physical contact with any surfaces present in the surroundings leads to the transfer of microorganisms from the human body to the surrounding surfaces. Floors are one of the most important surfaces for establishment and growth of such microbes because of presence of uneven areas and crevices that can hold moisture. [3] Most of the organisms present as common floor microflora, are opportunistic and can cause infections at higher concentration. For example, Serratia marcescens is a human pathogen and is responsible for catheter-associated bacteremia, urinary tract infections and wound infections [4], Staphylococcus aureus can cause minor skin infections such as pimples. [5] Pseudomonas aeruginosa typically infects the pulmonary tract, urinary tract, burns, wounds, etc. [6] Aspergillus versicolor can produce toxins, Cladosporium spp. cause infections of the skin, toe nails, sinusitis and pulmonary infections. Children, aged individuals and patients are relatively more susceptible to such infection because of their low levels of immunity. Earlier work in the laboratory has suggested that unhygienic places supported by moisture content facilitated maximum microbial growth. It is practically impossible to eradicate microorganisms from the floor. Generally it is accomplished using phenyl as a disinfectant and deodorant, its prolong use is not safe for the health as is made up of chemicals that may damage the skin and cause skin diseases. [7] With the increase in the knowledge and research in this field, disinfectants were introduced. Disinfectants are the best weapons for fighting against germs. It is an agent, such as heat, radiation, or a chemical, that destroys, neutralizes or inhibits the growth of microorganisms. [8] Different disinfectants are presently available commercially which have pleasant smell along with the ability to inhibit the growth of microorganisms. There are a number of manufacturers who have launched various disinfectants commercially in the market. There is cut through competition for the market share of Fast Moving Consumer Goods (FMCG). To www.wjpps.com Vol 6, Issue 3, 2017. 1317

have an edge over others, all are coming up with better and catchy advertising campaigns day by day. As a lay man, it is obvious to get biased and buy the disinfectants which have better packing and promotion campaign. Indian Medical Association (IMA) states, a good disinfectant should be capable of killing the germs by 99.99% within 60 seconds and recommends only Lizol and Dettol. 1.1 OBJECTIVES OF THE STUDY The present project is designed to study the efficiency of the floor disinfectants with the following objectives: 1. To conduct a survey on most frequently used floor cleaners by consumers. 2. To study the Efficacy tests of the floor cleaners at different concentrations by using turbidometric analysis. 3. To study the Longevity of floor cleaners in controlling microorganisms for 2 to 4 hours. MATERIALS AND METHODS The project was carried out in the following steps: 1. Consumer Survey for frequently used floor disinfectants 2. Efficacy tests of the floor cleaners at different concentrations 3. Study of Longevity of floor cleaners in controlling microorganisms 1. Consumer Survey for frequently used floor disinfectants The participants of the project conducted a basic survey on 200 consumers for the floor cleaners used by them. Most of the people are from urban area in and around Visakhapatnam. The format of the survey was mentioned as below: S.NO. DATE NAME OF THE CONSUMER FLOOR CLEANER USED NO. OF CLEANINGS PER DAY OR WEEK 2. Efficacy tests of the floor disinfectants at different concentrations Antiseptics and disinfectants are chemical substances used to prevent contamination and infection. Many are available commercially for disinfection and asepsis. Phenol controls by the germicidal effect caused by alternation of protein structure resulting in protein denaturation. It acts as surface-active agent (surfactant) precipitates cellular proteins and disrupts cell membranes. Usually 5% solutions are used for disinfection. Cresols are also having similar effect with 2 to 5% solutions. www.wjpps.com Vol 6, Issue 3, 2017. 1318

The efficiency of all disinfectants and antiseptics is influenced by a variety of factors, including the following: i. Concentration: The concentration of a chemical substance markedly influences its effect on microorganisms with higher concentrations producing a more rapid death. Concentration cannot be arbitrarily determined; the toxicity of the chemical to the tissues being treated and the damaging effect on non-living materials must also be considered. ii. Length of exposure: All microbes are not destroyed within the same exposure time. Sensitive forms are destroyed more rapidly than resistant ones. The longer the exposure to the agent, the greater its antimicrobial activity. The toxicity of the chemical and environmental conditions must be considered in assessing the length of time necessary for disinfection or asepsis. iii. Type of microbial population to be destroyed: Microorganisms vary in their susceptibility to destruction by chemicals. Bacterial spores are the most resistant than noncapsulated forms; acid-fast bacteria are more resistant than non-acid-fast and older; metabolically less active cells are more resistant than younger cells. Awareness of the types of microorganism that may be present will influence the choice of agent. iv. Environmental conditions such as temperature, ph and type of material on which the microorganisms exist. In the present study 5ml of sterilized nutrient broth was taken to test 7 various concentrations of disinfectants including the recommended and concentrated solution. 1 x 1 square feet area of floor was marked for this study. This area was wiped with sterile cotton to remove dust first and a sample was taken with swab dipped in saline water. The swab was dipped in 5ml of nutrient broth and kept aside under sterile condition. This sample was used as positive control. Then the same area was wiped with 0.15 µl/ml Lizol. After 10 minute area was wiped with swab dipped in saline water. The same process was repeated with different concentrations such as 0.6 µl,3 µl,15 µl, 60 µl, 240 µl and 600 µl concentrations of Lizol. In the same manner the samples were collected, using Dettol, Phenol and Domex. All the samples were incubated at 37 0 C for 24 hrs. The samples were measured for growth using Turbidometric analysis and the optical density (OD) was recorded at 470 nm using the colorimeter. Sterilized broth was used as negative control with each set. www.wjpps.com Vol 6, Issue 3, 2017. 1319

3. Study of Longevity of floor cleaners in controlling microorganisms To compare the longevity of effectiveness of the disinfectants another test was carried out for about 4 hours. Floor cleaners used in houses, hospitals and laboratories must be tested periodically to ascertain its potency and efficacy. Evaluation for concentration is not enough. But it is for the activity of the agent under certain conditions and uses. The concentration or dilution of the disinfectant to be tested may be based on manufacturer s recommendations. To confirm the findings samples were collected after every 2 hours interval after cleaning the floor with respective disinfectant using recommended concentration (Considered as 0 hour). The solutions should be prepared on the day of test. Lizol of 240, Dettol of 60, Domex of 240 and Phenol of 15 µl/ml concentrations were selected for this analysis. In this test 5ml of nutrient broth was taken in test tubes and sterilized. 1 x 1 square feet area of floor was marked for this study. This area was wiped with sterile cotton to remove dust first and a sample was taken with swab dipped in saline water. The swab was dipped in 5ml of nutrient broth and kept aside under sterile condition. This sample was used as positive control. Then the same area was wiped with Lizol. After 10 minute area was wiped with swab dipped in saline water. Another 2 samples were collected after 2 hours and 4 hours respectively. In the same manner the samples were collected, using Dettol, Phenol and Domex. All the samples were incubated at 37 0 C for 24 hrs. The samples were measured for growth using Turbidometric analysis and the optical density (OD) was recorded at 470 nm using the colorimeter. Sterilized broth was used as negative control with each set. RESULTS AND DISCUSSION It is practically impossible to keep our surroundings (including floor) free from microorganisms because they can grow in abundance in the presence of moisture. Cleaning of the floor will not be able to dislodge the microbes completely, but use of suitable disinfectant will help to decrease the growth of organisms to avoid potential infection that can be caused by higher potential load of the same pathogen. For the very reason the floor with which one is in direct contact needs to be cleaned regularly with suitable disinfectant and cleansers. The most frequently occurring bacterial species have been isolated and identified by using cultural & biochemical techniques. As per the previous studies it was revealed that, Escherichia coli, Micrococcus luteus, Serratia marcescens, Staphylococcus aureus, Pseudomonas aeruginosa & Bacillus cereus are predominantly present on floor. These organisms in less potential load do not cause infections rapidly. But when load increases due www.wjpps.com Vol 6, Issue 3, 2017. 1320

to uncontrolled growth i.e., because of uncleaned area chance of development of infections of diseases occur. Previous studies have used the floor disinfectant that are sold maximum and tried to compare their efficacy using in vitro analysis (Floor samples were collected and inoculated in the broth along with floor disinfectant and then incubated under controlled conditions). However, in practice, at places like home, laboratory etc. maintaining controlled conditions is impossible, therefore, to confirm the effect of floor disinfectant in vivo experiments were designed in the present study. In order to control the growth of dust and the microbial load people use floor cleaners such as Lizol, Dettol, Domex, Phenol and others. 3.1 Consumer Survey for frequently used floor disinfectants: The results of basic survey conducted amongst 200 consumers are represented in Figure 1. It revealed that Lizol, Domex and Dettol (antiseptic) are most frequently used products for domestic purpose. Majority of the clinics and hospitals prefer Dettol (antiseptic) as disinfectant. More than 90% of the people do not follow the standard concentration to be used, as recommended by the manufacturer. Figure 3.1. The results of basic survey conducted amongst 200 consumers People clean/swipe their floor once ( 88%) or at the most twice a day ( 12%) which indicated the importance of analysing the longevity of a particular disinfectant. People are happy with what they use and only a few are open to try new products launched. Instead of www.wjpps.com Vol 6, Issue 3, 2017. 1321

the composition and inhibition factor, to select a specific disinfectant, fragrance and cost are major criteria. With respect to the above stated information obtained by survey analysis and to confirm the earlier work done in our laboratory, in the present study commonly used floor disinfectants like Lizol, Domex, Dettol and Phenol are used. Earlier work suggests no difference in various methods like Agar Ditch, Paper Disk and Turbidometric analysis that can be used for these kinds of experiments. However, paper disk and agar ditch is difficult to perform with respect to floor micro flora as some of the samples need to be concentrated. Therefore, in the present study, to determine the effectiveness of products Turbidometric analysis is performed. 3.2 Efficacy tests of the floor cleaners at different concentrations The value of OD of negative control was subtracted from all other readings to nullify the effect of colour imparted by nutrient broth. The OD of positive control was considered as 100% growth and compared with OD obtained using test samples to calculate % growth of floor microorganisms after the use of particular disinfectant. To calculate % inhibition, the values of % growth was subtracted from 100 and plotted on the graph against concentration or time of incubation. 3.2.1 Effect of various concentrations of Lizol on microorganisms 10% inhibition of microbial growth was observed with 0.15µl/ml, 15% with 0.6 µl/ml, 24% with 3 µl/ml, 27% with 15 µl/ml, 35% with 60 µl/ml, 60% with 240 µl/ml and 80% with 600 µl/ml of Lizol. www.wjpps.com Vol 6, Issue 3, 2017. 1322

Figure. 3.2.1 represents the results obtained for % inhibition in the growth of floor microorganisms using in vivo analysis by turbidometric method using Lizol. 3.2.2 Effect of various concentrations of Dettol on microorganisms: 2% inhibition of microbial growth was observed with 0.15 µl/ml, 10% with 0.6 µl/ml, 24% with 3 µl/ml, 40% with 15 µl/ml, 50% with- 60 µl/ml, 55% with 240 µl/ml and 75% with 600 µl/ml of Dettol. Figure. 3.2.2 represents the results obtained for % inhibition in the growth of floor microorganisms using in vivo analysis by turbidometric method using Dettol. 3.2.3 Effect of various concentrations of Phenol on microorganisms 24% inhibition of microbial growth was observed with 0.15 µl/ml, 27% with 0.6 µl/ml, 45% with 3 µl/ml, 50% with 15 µl/ml, 55% with 60 µl/ml, 75% with 240 µl/ml and 95% with 600 µl/ml of Phenol. www.wjpps.com Vol 6, Issue 3, 2017. 1323

Figure. 3.2.3 represents the results obtained for % inhibition in the growth of floor microorganisms using in vivo analysis by turbidometric method using Phenol. 3.2.4 Effect of various concentrations of Phenol on microorganisms 7% of inhibition of microbial growth was observed with 0.15 µl/ml, 20% with 0.6 µl/ml, 26% with 3 µl/ml, 45% with 15 µl/ml, 48% with 60 µl/ml, 60% with 240 µl/ml and 90% with 600 µl/ml of Domex. Figure. 3.2.4 represents the results obtained for % inhibition in the growth of floor microorganisms using in vivo analysis by turbidometric method using Domex. www.wjpps.com Vol 6, Issue 3, 2017. 1324

3.3 Study of Longevity of floor cleaners in controlling microorganisms Manufacturers of all the disinfectants used in the present analysis recommend use of 3μl/ml of water. Most of the disinfectants used showed 25% inhibition in the growth of floor microbes with recommended concentration, except Phenyl, which is able to inhibit 47% and 44% growth respectively. Maximum inhibition (> 90%) was observed when concentrated samples were used for all the disinfectants. That means the recommended concentration by the manufacturer is not sufficient enough. These disinfectants are not able to inhibit the growth of floor microorganisms efficiently at recommended concentration. To determine comparative longevity of effect of floor disinfectants, the floor cleaner was applied to the floor and the samples were collected at 0 hours, 2hours and 4 hours. The results are represented as follows: 3.3.1 Effect of Lizol after 2 and 4 hours: 35% of inhibition in microbial growth was observed at 0 hours, 25% after 2 hours and 0% after 4 hours with 240 µl/ml of Lizol. Figure 3.3.1 Inhibition in growth of total floor microorganisms collected after every 2 hours after cleaning with standard concentration of Lizol. 3.3.2 Effect of Dettol after 2 and 4 hours: 30% of inhibition in microbial growth was observed at 0 hours, 25% after 2 hours and 8% after 4 hours with 60 µl/ml of Dettol. www.wjpps.com Vol 6, Issue 3, 2017. 1325

Figure 3.3.2 Inhibition in growth of total floor microorganisms collected after every 2 hours after cleaning with standard concentration of Dettol. 3.3.3 Effect of Phenol after 2 and 4 hours: 24% of inhibition in microbial growth was observed at 0 hours, 55% after 2 hours and 40% after 4 hours with 15 µl/ml of Phenol. Figure 3.3.3 Inhibition in growth of total floor microorganisms collected after every 2 hours after cleaning with standard concentration of Phenol. 3.3.4 Effect of Domex after 2 and 4 hours: 45% of inhibition in microbial growth was observed at 0 hours, 40% after 2 hours and 28% after 4 hours with 240 µl/ml of Domex. www.wjpps.com Vol 6, Issue 3, 2017. 1326

Figure 3.3.4 Inhibition in growth of total floor microorganisms collected after every 2 hours after cleaning with standard concentration of Domex. Finally Phenol was found to be effective by inhibiting 55% after 2 hours and after 4 hours also its effect was good with 40% of inhibition of microbial growth. CONCLUSIONS According to the survey Lizol, Domex and Dettol (antiseptic) were high on the charts, whereas, phenol is amongst less preferred brands as floor disinfectant. Lizol and Dettol are the disinfectants that are recommended by the Indian Medical Association [9], but from the above study it was found that Phenol showed maximum inhibition of microorganisms at the recommended concentration. Concentrated solution of Phenol gave maximum inhibition as compared to concentrated solutions of any other disinfectant used. Phenol also proves to be the one to give consistent and long lasting effect minimizing reoccurrence of the inhibited microbes. Domex proves to be more effective disinfectant as compared to Lizol after Phenol. The present study from our laboratory using various disinfectants with various concentrations and various time intervals suggests that Phenol is most efficient and long lasting amongst the floor cleansers used. Phenol s ability to inhibit growth of floor microorganisms is better than Lizol which is recommended by Indian Medical Association. We highly recommend use of Phenol as floor disinfectant. www.wjpps.com Vol 6, Issue 3, 2017. 1327

ACKNOWLEDGEMENTS The department of Microbiology of is highly thankful to the Principal, Dr. V.S. Krishna Govt. Degree College, Visakhapatnam for providing financial help and laboratory facilities. The department of Microbiology Honours The Commissioner, CCE, Andhra Pradesh for the support and encouragement given to the student study projects. REFERENCES 1. Nene YL. A glimpse at viral diseases in ancient period. Asian Agri-History, 2007; 11(1): 35-46. 2. Pelczar MJ, Chan ECS and Kreig NR. Microbiology- 5th Edition, New-York, Tata Mac Graw Hill Publishing Co. Ltd, 1993; 137-386. 3. www.wordnetweb.princeton.edu/perl/webwn. 4. Stainer RY, Ingraham JL, Wherlis ML and Painter PR. General microbiology- 5th edition, MacMillan Press Ltd, London. 2001; pp- 2. 5. Auwaerter P. "Serratia species". Point-of-Care Information Technology ABX Guide. Johns Hopkins University. 2008. 6. Daftary VG, Banker DD and Daftary GV. Clinical Microbiology-First Print, Bombay, Universal Publishing Corporation, 1996; 12-55. 7. www.healthand energy.com/aspergillis.htm. 8. Sinha AS, Dasani SH and Rai SA. Germ Invasion and a Comparative Analysis of various Floor Cleaners, Electronic Journal of Biology 2009; 5(1): 17-21. 9. www.lizol.co.in. www.wjpps.com Vol 6, Issue 3, 2017. 1328