Indian Journal of Ophthalmology

: 2003  |  Volume : 51  |  Issue : 2  |  Page : 171--176

Microbiological evaluation of various parameters in ophthalmic operating rooms. The need to establish guidelines.

U Kelkar, S Kelkar, Abhijit M Bal, S Kulkarni, S Kulkarni 
 Microtech Diagnostics, Pune, India

Correspondence Address:
U Kelkar
Microtech Diagnostics, Pune


Purpose: Postoperative infections can be caused by a contaminated environment, unsterile equipment, contaminated surfaces, and infected personnel as well as contaminated disinfectants. In order to establish guidelines for microbiological monitoring, a detailed microbiological surveillance was carried out in an ophthalmic hospital. Method: Over a period of 21 months, we assessed environmental Bacteria Carrying Particle (BCP) load and surface samples weekly (n=276); the autoclaving system once a month and repeated whenever the process failed (n= 24); the air conditioning filters for fungal growth once in four months (n = 15), and the disinfectant solution for contamination once in two months (n = 10). Additionally, the personnel involved directly in surgery were screened for potential pathogens such as Staphylococcus aureus and β haemolytic streptococci. Result: On 14 (5.07%) occasions the environment in the operating rooms had a significant risk of airborne infections. Sterilisation of instruments in the autoclaves was unsatisfactory on 4 (16.66 %) occasions. Samples from the filters of the air-conditioning units yielded potentially pathogenic fungi on 3 (20%) occasions. Personnel sampling revealed that 5 (8.77%) individuals harboured β haemolytic Streptococci in the throat and 4 (7.01 %) harboured S. aureus in the nasal cavity. The samples of disinfectant in use were not contaminated. Conclusion: There is a need to standardise microbiological evaluation protocols for operating rooms.

How to cite this article:
Kelkar U, Kelkar S, Bal AM, Kulkarni S, Kulkarni S. Microbiological evaluation of various parameters in ophthalmic operating rooms. The need to establish guidelines. Indian J Ophthalmol 2003;51:171-176

How to cite this URL:
Kelkar U, Kelkar S, Bal AM, Kulkarni S, Kulkarni S. Microbiological evaluation of various parameters in ophthalmic operating rooms. The need to establish guidelines. Indian J Ophthalmol [serial online] 2003 [cited 2022 Aug 12 ];51:171-176
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Full Text

Postoperative infection remains a major cause of morbidity amongst patients undergoing surgery. Maintenance of strict asepsis is essential if postoperative infections and their consequences are to be minimised. Such infections can be either endogenous or exogenous. Factors associated with transmission of infective material exogenously in a hospital are use of unsterile equipment, presence of shedders of pathogenic microorganisms amongst hospital personnel, contaminated environment and contaminated surfaces.[1]

The ophthalmic operating room (OR) is a highly specialised unit where strict asepsis needs to be maintained because of fear of postoperative infections including endophthalmitis.[2] The incidence of postoperative endophthalmitis sporadically or as mini-outbreaks especially in ophthalmic camps has increased. It is possible and feasible to prevent such infective complications with proper disinfection and sterilisation routines. To judge the overall efficacy of disinfection methods and sterilisation of instruments, a detailed microbiological surveillance was carried out in a hospital where only ophthalmic surgeries are performed. This also included screening the hospital personnel for shedding of pathogenic microorganisms. An attempt was made to identify critical areas where microbiological surveillance was necessary.

 Materials and Methods

The study was carried out at the National Institute of Ophthalmology, a specialty ophthalmic hospital and day care center in Pune, at weekly intervals from August 2000 to April 2002. The hospital has three ORs. The hospital routine for sterilisation of the OR consisted of (1) high-level disinfection every Saturday by formaldehyde gas generated by addition of KMnO 4 and 40% liquid formalin; (2) additional fumigation whenever an obviously infected eye was operated; and (3) daily ultraviolet irradiation for 12-16 hours before surgeries. The ORs were sealed off for 24-36 hours following formaldehyde fumigation before the next surgery. Liquid ammonia solution was used to neutralise the irritant effects of formaldehyde two hours before surgery.

Environmental Bacteria Carrying Particle (BCP) load by sedimentation method was assessed weekly following high-level disinfection and prior to commencement of surgery.[3] According to this method a 10-cm diameter blood agar plate was exposed to the operating room air for 30 minutes. During this period care was taken not to generate air currents in the room under evaluation. The blood agar settle plate was then incubated at 37C for 24 hours. BCP load in the environment was determined by a formula based on the colony count, area of the plate exposed, and the duration of exposure. The number of BCP settling on 1 m 2 of medium per minute is equal to the number of such particles per 0.3 cubic meter of air. Since the agar plate is 10 cm in diameter (i.e. 5 cm radius), its area (pr 2) is approximately equal to 78 cm 2, i.e., 0.0078, m 2. The acceptable upper limit of bacteria is 180 BCP/m. 2 This equals 54 BCP/0.3 m 2. Therefore, 1 m 2 of agar medium exposed for one minute should not have more than 54 colonies. Alternatively, 0.0078 m 2 exposed for 30 minutes should not yield more than 54 x 30 x 0.0078, i.e., 12.6 colonies. Making the acceptable limit more stringent, we fixed 10 instead of 12 as the upper limit for certifying the OR safe for surgery. Briefly, the number of bacteria carrying particles settling on 1 m 2 of medium per minute is equal to number of such particles per 0.3 cubic meter of air. Approximately, 180 bacteria per cubic meter of air correspond to 10 colonies settling on a plate. The operating rooms were said to be conducive for carrying out operative procedures only when the bacterial load was less than 180 per cubic meter.[4]

Qualitative analysis of the colonies was done using standard bacteriological methods[5] to rule out the presence of Staphylococcus aureus. Detection of even a single colony of S.aureus was considered a risk for infection.[4]

Surface swabs were collected from representative areas in the operating room. These included the operation table at the head end, overhead lamp, the wall near the electrical switches and the floor below the head end of the table. The swabs were inoculated in Robertson's cooked meat media. After incubation at 37C for 4 hours, anaerobic cultures were subsequently undertaken on blood agar plates. These samples were also collected weekly from each OR.

Swabs were collected from filters of air-conditioning units and streaked on Sabouraud's dextrose agar without antibiotics to isolate fungi. A sampling interval of four months was maintained. A total of 15 evaluations were performed from the three air-conditioning units, five times from each unit. If the cultures yielded fungi, the air conditioner filter was meticulously cleaned using a sporicidal disinfectant such as chlorine dioxide in adequate concentration.

To determine the efficacy of sterilisation by autoclaves, commercially available spore strips (Hi-media, Mumbai) impregnated with 10[5] spores of Bacillus sterothermophillus were used. Spore strips were inserted into the cold compartment of the autoclave, which is the lowest part of the chamber.[6] Strips were processed as per the standard protocol.[6] After autoclaving of the load, the strips were aseptically transferred in trypticase soy broth which was incubated at 56C for five days as per the established methods. The broth was examined intermittently for signs of turbidity. The efficacy of the autoclaving process was checked on a monthly basis and the evaluation repeated whenever the process failed. A total of 24 evaluations were performed.

With written informed consent, personnel directly involved in patient care such as the operating and assisting surgeons, assistant surgeons, anesthesiologists and OR nurses were screened for presence of S.aureus from the anterior nasal vestibules and for β haemolytic streptococci from the posterior pharyngeal wall. Swabs from these areas were plated on suitable selective media, namely, salt milk agar for S.aureus and crystal violet blood agar for β haemolytic streptococci. Bacteria were identified using standard bacteriological techniques.[5] Since seasonal changes are known to increase the incidence of respiratory tract infections this evaluation was carried out twice a year prior to onset of rains and around winter. During the study period, 16 personnel were evaluated three times. The OR personnel detected to harbor S.aureus were treated with an intranasal application of mupirocin ointment along with chlorhexidine hair wash and use of medicated soap for seven days. Those found to harbor β haemolytic streptococci were administered 500 mg of oral amoxicillin three times a day for five days. Sampling was repeated after treatment. Thus, a total of 57 evaluations were done.

Savlon (0.6% Cetrimide and 0.3% Chlorhexidine gluconate) diluted 1:3 in water was used as a general-purpose disinfectant solution in the hospital. The in-use disinfectant bottle with chetals forceps was examined for any contamination. Briefly, 1ml of disinfectant solution was added to 9 ml of nutrient broth with tween 80-3% w/v and 10 drops from this solution were inoculated separately on two nutrient agar plates. These plates were then incubated, one at 37C for 3 days and the second at room temperature for 7 days. The disinfectant was considered unsatisfactory when 5 or more colonies were grown on either plate. Evaluation for contamination was done once in two months.


The 3 operating rooms were monitored for 92 consecutive weeks from August 2000 to April 2002. Taken together, the ORs were evaluated on 276 occasions for environmental BCP load and surface sampling for the presence of Clostridia. On 14 (5.07%) occasions the environment in the OR had significant risk of airborne infections. This was due to presence of unacceptably high bacterial count on 9 (3.26 %) occasions and the presence of S.aureus on 5 (1.81 %) occasions as evidenced by examination of the settle plates. The OR surfaces were free of anaerobic contamination post fumigation on all except 2 (0.72 %) occasions. The details of the individual ORs are shown in [Table 1].

Sterilisation of instruments and drapes in the autoclaves was assessed using biological monitors 24 times during the study period. The sterilisation process was unsatisfactory on 4 (16.66 %) occasions. The swabs from the filters of the AC units were analysed for presence of fungi 15 times and on three occasions (20%) they were found contaminated with fungi. Fungi isolated were Mucor species twice and Aspergillus niger once. The personnel sampling surveys in all included 16 persons who worked in close association with the patients. A total of 57 evaluations were done. Five (8.77%) individuals harbored β haemolytic streptococci in the throat and 4 (7.01%) harboured S. aureus in the nasal cavity. None harboured both organisms simultaneously. These personnel were advised suitable treatment and during the period of treatment they were advised not to work in the OR. Samples collected and analysed after adequate treatment did not yield organisms. Posttreatment sampling was done only once. None of the personnel was detected as a carrier on more than one occasion.

The in-use disinfectant solutions in the OR were assessed on 10 occasions. The disinfectants were never found unsatisfactory for use.


Operating room layouts, operating room etiquette, sterilisation of instruments, and sterile surgical protocols are factors that directly affect the incidence of postoperative infections. Of all the processes the environmental disinfection and instrument sterilisation probably need the most critical monitoring. Periodic and regular microbiological surveillance of the AC units, the disinfectants in use and the personnel working in the vicinity during surgery is an additional need.

It has been observed that there is a general relationship between aerobic bacterial count and risk of infection. The risk of infection increases with counts in the range of 700-1800 BCP per cubic meter, and decreases when the BCP load is less than 180 per cubic meter.[4] In the present study, the BCP load was high, and S.aureus was present in the OR on 14 (5.07%) occasions. The hospital under study was not using laminar air flow systems for air filtration. The use of laminar air flow systems is advised in the OR though their value has not yet been documented for ophthalmic ORs. Laminar flow ventilation is unidirectional, delivering air flow of 300 air changes per hour over the operating table. Air is drawn in from the atmosphere and passes through a 5 m filter of 95% efficiency. Before the air is delivered to the operating site it passes through a 0.3m high efficiency particulate air (HEPA) filter with 99.97 % efficiency. The value of laminar flow for surgeries other than orthopedic surgery has been identified as an area for future research and for development of guidelines by the Centers for Disease Control and Prevention.[7]

In the ORs where planar ventilation systems are operative and infection control specialists monitor quality assurance, surface swabs are often not indicated. The utility of the bacteriological assessment of the surfaces of the ORs for the presence of Clostridia is questionable. In our study Clostridia were isolated only twice out of 276 evaluations. At best it is an indicator of effective cleaning and housekeeping practices. However, the value of this tool for sentinel surveillance is good.

The biological indicators are recognised as the best monitors for monitoring sterilisation process by autoclave because unlike chemical indicators, they assess the sterilisation process directly by using resistant forms of organisms ( B. sterothermophillus spores) and not by merely assessing the physical and chemical conditions necessary for sterilisation. The B. sterothermophillus spores used in the biological indicators are more resistant and present in greater numbers than the common microbial contaminants found on patient care equipment; hence the demonstration of inactivation of the biological indicator implies that other potential pathogens have been also killed during the sterilisation cycle.[8] In the present study, faulty pressure gauges, overloading and inadequate exposure time could be the possible causes of inadequate sterilisation on 4 occasions.

Healthcare associated infections attributable to fungi have been documented. Aspergillus is ubiquitous and occurs in soil, water and decaying vegetations. Other opportunistic fungi associated with nosocomial infections are Rhizopus, Fusarium and Penicillium.[9] All these are capable of proliferating in wall mounted air conditioning units. In fact, wall-mounted air conditioners are installed more for comfort than for clean air delivery and should not be used as air delivery systems. The units are usually mounted on the outside wall, and the air is invariably directed down and back onto itself towards the wall.[10] The filters utilized in these units can act as nidus for growth and proliferation of the pathogenic fungi. In the present study, filters of wall mounted AC units were found to harbour potentially pathogenic fungi on 3 (20%) occasions.

S.aureus and β haemolytic streptococci are important healthcare associated pathogens and can persist in the environment for extended periods. They can be shed and infections can occur when health care personnel are heavily colonized with these organisms. S.aureus and β haemolytic streptococci have been linked to airborne transmission in operating rooms, burn units, and neonatal units.[11], [12] In this study 4 healthcare personnel harboured S.aureus and 5 harboured β haemolytic streptococci . Periodic screening, treatment of shedders and abstaining from surgeries till the completion of treatment would ensure that the risk of transmission of infections is minimized.

Disinfectants in use in hospitals should always be freshly prepared and should be of adequate strength. Although the disinfectants in use were never found contaminated in this study, the number of samples was too small to comment upon. Nosocomial infections due to usage of contaminated antiseptics have been reported on numerous occasions. In a recent study carried out in Pune, India, antiseptic solutions in a large general hospital were found contaminated with pathogenic bacteria including Pseudomonas aeruginosa and Klebsiella species.[13]

Surveillance is essential for recognising nosocomial infection problems and for instituting effective preventive measures.[14],[15] In developed countries organisations such as the American Hospital Association and the Joint Commission on Accrediation of Healthcare Organisations (JCAHO)[16],[17] set standards of care within hospitals. Survelliance limited to monitoring of the patients while they are in hospital is not likely to detect infections that become evident only after the patient's discharge from the hospital.[18] Prospective studies have shown that invasive devices play a far more important role in determining susceptibility to nosocomial infections than the underlying disease.[19] Most patients undergoing ophthalmic surgery do not have underlying disease and hence the need for evaluation of all the possible potential sources which can lead to nosocomial infection in these patients assumes greater importance.

The various parameters studied by us are potential sources of infection if the available standards pertaining to each are not adhered to. We found that the BCP load in the environment was unacceptably high on more than 3% occasions, the steam sterilisation process failed on nearly 17% occasions and shedders of S.aureus and β haemolytic streptococci were detected in the OR personnel. The correlation between these findings and postoperative infections could not be established directly as precautionary corrective measures were undertaken immediately.

Although specific guidelines about ophthalmic ORs are not available, hospitals should adhere to the definition of a clean room. A clear room is defined as one constructed and used in a manner to minimise introduction, generation and retention of particles inside the room and in which other comfort parameters such as temperature, humidity, and pressure are controlled. The required standard of cleanliness of a room depends on its use. A class 100 clean room is required for implant or transplant surgeries, isolation of immunosuppressed patients, (e.g. after bone marrow transplant operations) and also when a bacteria-free or particulate-free environment is required (e.g. manufacture of aseptically-produced injectable medicines).

Operating rooms for ophthalmologic surgeries must abide by certain physical and microbiological parameters so as to ensure that the chances of exogenous postoperative infections are minimised. Physical parameters based on the guidelines for design and construction of hospitals and healthcare facilities[20] are summarised in [Table 2]. As per these guidelines there should be a minimum of 15 total air changes per hour, a relative humidity of 30-60% and a design temperature of 20-30C.

The microbiological parameters are less well defined [Table 3]. We suggest that biological indicators assessment should be carried out at monthly intervals, environmental BCP load evaluation at weekly intervals, and evaluation of OR staff for carriage of S.aureus and β haemolytic streptococci twice in a year (in the months of June and December). Monitoring of AC units for fungi is an additional infection control measure. A monitoring frequency of about 3 to 12 evaluations per year is adequate. Disinfectants should ideally be reconstituted to adequate dilutions centrally in a hospital and then distributed to different locations with a label indicating date of reconstitution.

In India there is a need to develop standard guidelines for monitoring ophthalmic operating rooms. The present study was carried out in only one eye hospital in India and there is need to conduct similar prospective studies of sufficient duration in other hospitals before a set of national guidelines can be established.

An ideal infection control protocol would also consist of educational programs for operating room personnel through seminars and audiovisual teaching aids.


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