The bacterial strains, obtained from lyophilized culture collection at the Adolfo Lutz Institute (IAL, SP, Brazil), were Enterobacter cloacae IAL 1976; Serratia marcescens IAL 1478; and Acinetobacter calcoaceticus ATCC 19606, IAL 124. The reference bacteria used were Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Bacillus subtilis var. globigii ATCC 9372, and Bacillus stearothermophilus ATCC 7953.

Working cultures were kept on Tryptic Soy Agar (TSA, Difco, Detroit, Michigan, USA) at 4°C with weekly transfers. The 24 h cultures, grown on TSA at 22°C for S. marcescens, and at 35–37°C for E. cloacae, A. calcoaceticus, E. coli; S. aureus, were harvested into Tryptic Soy Broth (TSB, Difco), centrifuged (1000 × g/ 15 min/ 4°C) and suspended in saline solution (0.9% NaCl). Bacterial viability was estimated on TSA pour plates by confirming populations ≥ 10⁶ CFU/mL. Spore cultures were developed for a period of six days in a sporulation medium [g/L: D-glucose, 2.5 (Sigma, St. Louis, Missouri, USA); L-glutamic acid, 0.4 (Sigma); yeast extract, 4.0 (Difco); peptone, 5.0 (Difco); sodium chloride, 0.01; manganese sulfate, 0.01; bacteriological agar, 20.0 (Difco)] at 37°C for B. subtilis, and at 62°C for B. stearothermophilus were harvested, centrifuged (4 times at 1935 × g/ 30 min), and kept suspended in chilled 0.02 M calcium acetate solution (pH = 9.7) at 4°C [15]. The viability of heat-shocked (80°C/10 min for B. subtilis and 100°C/ 20 min for B. stearothermophilus) spores was obtained through TSA pour plates by confirming populations ≥ 10⁶ spores/mL. The spore suspensions of B. subtilis and B. stearothermophilus were used for the tests of the D-value determination.

Chlorhexidine digluconate (biguanide, 1,6-dichorophenyldiguanido hexane; 40% w/v; Zeneca Farmacêutica, SP, Br); and sodium dichloroisocyanurate (NaDCC, sodium salt 50% w/w in tablets, Johnson and Johnson, J&J, SP, Br), glutaraldehyde (1,5-pentanediol; 2.0% w/v with sodium bicarbonate, pH = 8.3, Aster Produtos Médicos, SP, Br), formaldehyde (monoaldehyde; 37% w/v, Aster Produtos Médicos, SP, Br); sodium hypochlorite (10% w/v, Aster Produtos Médicos, SP, Br); hydrogen peroxide (40%, Laborosa Farmacêutica, SP, Br); a mixture of peracetic acid (4.5% v/v, PAA) and hydrogen peroxide (2.2% v/v, H₂O₂) plus acetic acid, 10 mg/L (Minncare^®, pH = 1.3, Minntech Corporation, Minneapolis, MN, USA) were used. The chemical solutions were prepared in sterile water for injection (WFI) to obtain: 0.4% and 2.0% v/v Chlorhexidine (pH = 6.2); 2.0% w/v glutaraldehyde (pH = 7.4); 0.1% and 0.2% v/v sodium dichloroisocyanurate (pH = 7.0); 0.025%, 0.05% and 0.1% sodium hypochlorite (pH = 7.0); 0.5% v/v formaldehyde (pH = 6.5); 1.0% v/v Minncare^® (0.45% of PAA + 2.2% of H₂O₂, pH between 2.0 and 2.3) and 1.5% v/v and 26.5% hydrogen peroxide (pH = 3.3). The concentration of total available chlorine and hydrogen peroxide was determined by the iodometric method [16]. The dilute solutions, prepared with chlorine-free glassware, were filtered through a 0.22 μm membrane (Millipore, Bedford, MA, USA) for daily use. The solutions of the test chemical agents are approved for hospital disinfection use by the Brazilian Ministry of Health [11].

The agents [17] used to inactivate the test disinfectants in solutions at 1% concentration were, respectively, polysorbate 80 (Tween 80) to quench chlorhexidine, glycine to bind glutaraldehyde and formaldehyde, catalase to degrade peracetic acid plus hydrogen peroxide; sodium thiosulphate to degrade sodium dichloroisocyanurate and hypochlorite. The efficacy of the disinfectant inactivating agents were confirmed as follows: (i) not having an inhibitory effect on the previous bioburden; (ii) completely eliminating the activity of the disinfectant; (iii) and when combined with the disinfectant, the resultant product was non-toxic to the test bacterium.

The resistance value (death kinetics) of a BI was characterized in terms of decimal reduction time, D-value, which is the exposure time required, under specified set of conditions, to cause one log₁₀ or 90% reduction of the initial population (N₀, bioburden) of viable BI in the suspension [7,8]. The determination of the D-value for the tested agent consisted in the transference of 1.0 mL of a 24 h suspension of the bacterial strain into 100 mL of the disinfectant solution that was kept in constant agitation at a controlled temperature room (at 25°C ± 1.0°C), at time zero. The initial concentration of bacteria (N_o) exposed to the disinfectant at time zero was around 10⁴ to 10⁵ CFU/mL (colony forming units/mL). At regular intervals (1 min for vegetative forms and 5 min for spore forms), a sample of the 1.0 mL mixture was transferred to 8 mL of TSB containing 1 mL of an inactivating agent at 1% concentration to guarantee a complete inactivation of the disinfectant without interfering with survivor growth. Using TSA pour plates, the survivors were evaluated by dilution (1:10, 1:100, 1:10³, 1:10⁴, 1:10⁵) in saline solution. A negative control was made in a tube of 9 mL TSB plus 1 mL of an inactivating agent. A positive control was made adding 0.1 mL organism suspension in 10 mL TSB in a tube. The method for each disinfectant and test bacterium was repeated at least four times, when bacterial cultures were exposed to four different samples of diluted disinfectant, before the determination of D-values by the survivor curves.

The vegetative strains which showed the best resistance to the solution of 0.4% chlorhexidine were E. cloacae and S. aureus, at least 1.5 times greater than the D-values shown by the more sensitive ones, which were A. calcoaceticus, S. marcencens and E. coli. A time interval of 3 to 4 min was enough to reduce 90% of the population of E. coli, S. marcescens and A. calcoaceticus; a 3log₁₀ reduction in bioburden for these species varied between 9 to 12 min.

The spore strains exposed to 2.0% chlorhexidine showed D-values 1.4 times higher for B. stearothermophilus than for B. subtilis.

It was observed that the strongest 2.0% chlorhexidine solution (5x that concentration of 0.4% chlorhexidine used against the other bacteria) showed a positive activity against populations of B. subtilis and B. stearothermophilus. It is unusual to find that alcohol/chlorhexidine mixtures would show any activity against spores, and may reflect log reductions of vegetative cells that remain in the spore population used for testing purposes. Therefore, the decimal reduction times reflected indeed log reductions of mixed populations formed of vegetative cells and spores, and this finding should be confirmed.

Chlorhexidine at 0.4% concentration in water or 70% alcohol is widely applied as a skin low-level disinfectant, becoming a leading hospital antiseptic in recent years due to its confirmed bactericidal (no sporocidal) effect and non-toxic side effects [11,20,21]. Chlorhexidine at 0.2% concentration is a well-known antimicrobial in the dental profession and 2.0% chlorhexidine solution has been applied to a disinfection of orthodontic prosthesis after mechanical cleansing.

In Brazil, 0.4%, 2.0% and 4.0% chlorhexidine solutions [11] are well-known as widely used disinfectants in hospitals. Within its principal applications, chlorhexidine is recommended for sanitizing the hands and the forearms of the surgical team and the patient's skin (pre-operative and invasive procedures), as well as bathing newborns. It was reported that a preparation with 0.5% chlorhexidine in 70% alcohol applied to baby skin before invasive procedures provided a significant delay in the colonization of catheters [20].

The 2.0% and 4.0% chlorhexidine used for surgical hand scrub products achieved a 3log₁₀ reduction in microorganisms from baseline population count, and the 2.0% solution caused less irritation to hands than the 4% preparation [20], considering 20 seconds of hands friction [11,21].

For total immersion of invasive medical devices, a total exposure time for: (i) 6 to 12log₁₀ reductions of vegetative bioburden with 0.4% chlorhexidine solution varied between 49.8 min and 99.6 min in relation to E. cloacae resistance, considering low disinfection level; and (ii) 12log₁₀ of sporulating strains (n = 12) varied between 80.4 min (1 h 20 min) and 109.2 min (1 h 49 min), considering high level disinfection with 2.0% chlorhexidine solution.

The 2.0% chlorhexidine in 70% alcohol will be tested in our laboratory to determine the D-values of the test strains of bacteria, as well as in the hand asepsis and the immersion of various medical devices at a hospital setting in the city of São Paulo.

In a solution of 5000 mg/L (0.5%) of formaldehyde, the spore formers B. stearothermophilus and B. subtilis evidenced similar D-values, respectively, showing twice the resistance of A. calcoaceticus and E. cloacae; and five-fold resistance as determined for S. marcescens, the most sensitive species.

Despite its effectiveness in low concentrations (0.5%), formaldehyde is a highly corrosive and toxic compound considered potentially carcinogenic. Its vapors irritate the eyes and lungs. A maximum concentration of 1.0 mg/L in the working environment is recommended by the National Institute for Occupational Safety and Health. Nowadays, the 37% formaldehyde concentrate solution is industrially prepared without any exhaling odor or irritation during handling but with all safeness. In Brazil, formaldehyde is less costly than glutaraldehyde, being selected to be used mainly in public health care centers for multiple purposes, under severe regulations [4,11].

In Brazil, in the hospital environment 4.0% formaldehyde solution is considered a high level disinfectant and is applied to a 24-hour exposure in the disinfection of capillary tubes of dialysis systems. For the semi-critical articles high level disinfection, formaldehyde in alcoholic at 8.0% v/v or aqueous at 10.0% v/v preparations are applied to an 18-hour exposure. Considering the low disinfection level of hospital devices, a 4.0% formaldehyde solution, for a period from 30 minutes to 4-hours, is suggested [11]. The recommended concentration of formaldehyde in preparations is 10 times higher than the concentration of the test formaldehyde solution at 0.5% that showed 12log₁₀ reductions of spores in a shorter time than that suggested by the Brazilian legislation. Both B. subtilis and B. stearothermophilus showed to be adequate BIs in the evaluation of the efficacy of formaldehyde in the immersion of medical devices. Periods from 65.4 min to 141.3 min are needed to reduce 6 and 12log₁₀ of the initial spore population, respectively.

Even for manufacturers of parenteral solutions, a solution of 1.0% formaldehyde has also been historically associated with disinfection of the water purification system. Recently, for the same applications, formaldehyde has been replaced by solutions of peracetic acid, with or without hydrogen peroxide [22].

In a solution of 2.0% glutaraldehyde, both spore formers B. subtilis and B. stearothermophilus showed the same D-value, D = 25.0 min, which is approximately 3 to 4 times greater than that determined for the most resistant vegetative species of E. coli and E. cloacae. The most sensitive vegetative strains were A. calcoaceticus, S. marcescens, S. aureus and E. cloacae.

The extensive use of the high level disinfectant glutaraldehyde is due to its special characteristics. It is active in the presence of organic material, and unreactive in natural and synthetic materials and detergents. It does not coagulate proteinaceous material, and it has no corrosive effects on metals and rubber. However, glutaraldehyde is a toxic compound, which irritates the skin, mucous and eyes. For this reason, as most chemical solutions, its handling demands the use of equipment for individual protection [4,11].

Glutaraldehyde is currently the most widely used chemical for high and medium levels of disinfection. A solution of glutaraldehyde is widely used in thermo labile and semi-critical articles, such as materials used in respiratory therapy, fiber optic endoscopes, as well as equipment used in dispensing anesthesia gas, metallic articles and suction equipment [23,24]. The Brazilian Legislature recommends the use of 2.0% glutaraldehyde for a period of 8 to 10-hours exposure for sterilization procedures [4,11].

B. subtilis and B. stearothermophilus proved to be BIs appropriate for the evaluation of glutaraldehyde as a sterilizing agent, requiring exposure times of 300 min (5 h), to undergo a 12log₁₀ reduction in viable spore forms. Glutaraldehyde is also appropriate in the disinfection of semi-critical articles, requiring 150 min exposure for a 6log₁₀ reduction, respectively, in populations of B. subtilis and B. stearothermophilus. Comparing the sterilizing effect of 0.5% formaldehyde and 2.0% of glutaraldehyde, the necessary time for decay n = 12 cycles of B. subtilis varied from 141.6 min (2 h 22 min) to 300.0 min (5 h), respectively. The 0.5% formaldehyde solution was more efficient in relation to that of 2.0% glutaraldehyde. The ability of glutaraldehyde to reduce n = 6 cycles of the vegetative strains varied from 28.2 min (A. calcoaceticus) to 42.6 min (E. coli) exposure time. After meticulous cleaning, exposing endoscopes for 120 min, was deemed to be sufficient for their immediate reuse.

The most resistant bacteria to the solution of 1.0% Minncare (0.45% peracetic acid plus 2.2% of hydrogen peroxide) were B. stearothermophilus, B. subtilis and E. coli, which showed 2–3 times higher D-values than the more sensitive strains of A. calcoaceticus, E. cloacae and S. aureus, which presented similar resistance.

The D-values evaluated for vegetative bacteria in 1.5% H₂O₂ (pH = 3.3) were similar to those determined in 1.0% Minncare mixture (pH = 2.3) with 2.2% H₂O₂. However, the presence of 0.45% peracetic acid provided to the mixture sporocidal activity, reducing 10 times the D-value of B. subtilis (from D = 55.2 min in 1.5% H₂O₂ solution to 5.9 min in 1% Minncare mixture). Considering a spore suspension of B. stearothermophilus, the D-value was 4.7 min for 26.5% H₂O₂, the concentration of which was 12 times greater than that present in 1% Minncare mixture. Peracetic acid at concentration variable ≤ 1.0% was confirmed sporocidal [1,25].

Minncare is less malodorous than glutaraldehyde, but it can irritate the eyes and respiratory tract requiring the use of personal protective gear during handling. A balanced mixture of peracetic acid, hydrogen peroxide and water decomposes to acetic acid and water and this is considered safe by most occupational safety guidelines. Its corrosive effects on metallic surfaces in high concentrations is a disadvantage. Minncare should not be used on materials like copper or bronze, but it is well compatible with plastics. Dilution and the addition of an anticorrosive agent convert it to a suitable product for the disinfection of endoscopes [25]. The recommended concentration for high level disinfection is a stabilized mixture of 0.2 to 0.35% peracetic acid and 4–6% hydrogen peroxide [1,25].

The stabilized 1.0% Minncare mixture can be applied to the cleaning of reverse osmosis membranes and continuous deionization apparatus during three hours to obtain purified water and 18-hours to obtain water for injection, which will be used to prepare parenteral solutions, including peritoneal dialysis solutions. According to Vessoni Penna et al. [22], strains of E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 showed the greatest resistance among the test vegetative Gram negative bacteria, when exposed, respectively, to 4.0% hydrogen peroxide and 1.0% Minncare (peracetic acid (0.45%) + hydrogen peroxide (2.2%) solution), suggesting that P. aeruginosa and E. coli should be both required for monitoring disinfection of the systems for water purification purposes.

In the recent work, B. stearothermophilus showed to be a more appropriate BI for the evaluation of peracetic acid and hydrogen peroxide solutions. For the reduction of 6 (n = 6 cycles) and 12 (n = 12 cycles) log₁₀ in viable counts, respectively, the effective exposure times were 54.6 min and 109.2 min. Among the vegetative strains, E. coli showed to be an adequate BI with a logarithm reduction factor of 6 in 40.2 min in comparison to 35.4 minutes observed for B. subtilis, for either disinfection of osmose reverse membranes (in water purification systems) or semi-critical articles in health care environment.

Hydrogen peroxide (H₂O₂) solution (1%–2% by weight), which has been used as a powerful oxidizing agent of proteins and other organic and inorganic encrustrated soils, is recommended for the low disinfection [11] of drinking water, non critical items, hard and smooth surfaces. A H₂O₂ solution at 1.5 ± 0.5% was prepared according to Brazilian hospital practices for cleaning medical devices [26], the concentration of which is easy to handle, has low toxicity and brings no damage to fiber materials and metal surfaces.

The most resistant vegetative strains to a 1000 mg/L (0.1%) solution of sodium dichloroisocyanurate (NaDCC) were A. calcoaceticus and E. coli, which showed the same D-value. The most sensitive vegetative strains, with similar D-values, were S. marcescens, E. cloacae and S. aureus. To reduce a logarithmic cycle of B. subtilis and B. stearothermophilus, an exposure time between 3.8 min and 4.4 min to 0.2% NaDCC was required.

The D-values for the vegetative test bacteria in 0.025% (250 mg/L) sodium hypochlorite (NaOCL) solution (pH = 7.0) ranged from 4.7 min for S. aureus to 7.5 min for both E. cloacae and E. coli, the D-values of which were close to those determined for the same strains exposed to 0.1% NaDCC solution. The resistance observed for the spore forms: B. subtilis (D = 20.6 min) and B. stearothermophilus (D = 24.0 min) exposed to 0.025% sodium hypochlorite were close and doubled the D-values achieved for the same spore forms, respectively, D = 10.3 min and D = 9.4 min, exposed to 0.05% sodium hypochlorite solution at the same conditions (pH = 7.0, 25°C).

Chlorine-based disinfectants react readily with organic material, including blood, excrement and tissues. Its antimicrobial activity is proportionally decreased in relation to the amount of the organic material present. Therefore, the concentration of chlorine available at the disinfection process should be sufficiently high to satisfy the expenditure of chlorine (chorine reacted with the organic material) and provide enough chlorine for anti-microbial activity.

The non-dissociated form, hypochlorous acid (HOCL) in water at pH 5–8, is responsible for the microbial inactivation by the chlorine releasing agents (CRAs) [25]. The dissociated forms, hypochlorite ions (OCl^-) that predominate in strong alkaline (pH>9.0) solutions, show less antimicrobial activity. At higher concentrations, the sodium hypochlorite (NaOCL) solutions are at pH>8 making the liberation of the active compound difficult [14]. NaOCL solutions, though effective at very low concentrations, as much as 250 mg/L (0.025%), as compared to the concentration of 1000 mg/L (0.1%) of NaDCC solution, have highly corrosive effects on metallic surfaces and irritate the skin [4,11,19].

The organic chlorine (NaDCC) can be used at higher concentrations than inorganic compounds (NaOCL) since NaDCC shows slow decomposition and liberation of active chlorine (HOCL). Its stability at pH 7 provides for exceptional experimental reproducibility. On the other hand, the 0.1% NaDCC solution shows lower toxicity, irritation to eyes and skin, less corrosive effects on metallic surface and less aggressiveness to plastic and rubber items than a solution of 250 mg/L of sodium hypochlorite.

In practice, NaDCC in the form of tablets, which dissolved very slowly and thereby released hypochlorous acid at the same rate it was being used, maintained an appropriate level of available chlorine without affecting the pH of the water [27]. Therefore, 0.1% NaDCC was recommended to be used in opened containers for intermediate disinfection level of semi-critical items; and 0.2% NaDCC solution might be applied to attain a high disinfection level at health care centers.

The recommendation of the Brazilian Ministerial directives [4,11] for the low level disinfection of lactates, inalotherapy, and oxigentherapy is an exposure of 60 min to a solution of sodium hypochlorite (NaOCL) at 250 mg/L (0.025%). Solutions of 0.5% NaOCL, for overnight exposure, are used to provide intermediate disinfection level of oxygen therapy and anesthesia devices, in hospital centers. Solutions of 1% NaOCL are applied to hard-surface disinfection of critical areas in hospital environments (intensive therapy rooms, nurseries, wards, operating theaters, special procedure places).

E. coli and A. calcoaceticus might have been used as appropriate BI for the evaluation of NaDCC as a disinfectant agent (n = 6 cycles) for an interval of 35.4 min. The high level disinfection parameter, n = 12, required exposure times of 53.3 min and 45.0 min for B. stearothermophilus and B. subtilis, respectively. B. stearothermophilus spores were observed to form appropriate BI to monitor sterilizing activity by a 0.2% NaDCC application.