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Microbiological Surveillance of Operation Theatre : Why… What…How …Where…Which…?” S. Poongodi @lakshmi, N. Palaniappan, M.Kannan, S.Nithya gomatheeswari

Abstract Purpose: This study was done to analyze the frequency, extent and methods of microbiological surveillance of OT, efficiency, microbicidal activity  of  product X, advantages of special air filters, air quality , rate and   factors contributing to SSI . Materials & methods:   Surveillance was done in two conventional OTs (GH) and two with special air filters(private) by settle plate, air sampler and surface swab for aerobe & anaerobe, from day 1- 6 in the morning & evening , after formalin and product X.  Microbicidal activity of product X was tested against common isolates. Results:       By settle plate or air sampler, the cfu were less with product X compared to formalin, the M- cfu were less than E- cfu and the cfu in the private OTs were less compared to GH. There was no gradual decrease or increase of cfu from day 1 to 6.Organisms isolated  were Acinetobacter baumanii, CoNS, MSSA and  micrococci in GH,  aerobic spore forming bacilli and  micrococci in private. RCM was negative for Clostridium tetani in all four OTs.  SSI was 14% in GH and Nil in private. Microbicidal activity of product X was good at 1% concentration within 10 mts. Conclusion: Air sampler measures the microbial burden more  accurately..  Settle plate is a direct indicator of SSI risk.  Clostridium tetani as an indicator of air quality may be limited to special situations only.. Peptone water swabs indicate the seriousness of the contaminant. Newer less toxic disinfectants are alternative to formalin. Correlation between microbial burden and SSI by any method is not absolute.

Keywords: OT, surveillance, method, disinfectant, SSI



Good hospital hygiene is an integral part of infection control programme. “Microbiological surveillance” provides data about the factors contributing   to infection. In developed countries, though standard protocols and guidelines are available, controversies over the frequency and extent of surveillance of Operation Theatres (OT) exist. In developing countries like India, where there is no uniform guidelines, many OTs are built and maintained  according to the individual’s knowledge level, availability of funds, technical staff, and  equipments.  The  validity  of  looking for Clostridium tetani spores in theatre swabs is  questionable .1 Bacterial air count is a suitable index in predicting post operative  infections  but not cost effective.2,3 Choosing the effective  and eco friendly alternative to formalin is  another need of the day.  Anything suggested for developed world may not be suitable to us unless studied and tailor made to resource poor settings.4


To study

– the three surveillance methods – settle plate, air sampler  and surface swabs (aerobic &    anaerobic)

-the efficiency of  product X, a commercial  disinfectant (balanced and stabilized      blend of per oxygen compounds, surfactant, organic acids & an inorganic buffer system) against 40% formaldehyde

-the advantages of special air flow arrangement over conventional OTs by studying the trend and quality of air between two subsequent fumigations

-the relationship between the quality of air in the OT and the rate of  surgical site infection (SSI)

-the factors contributing to SSI such as  type of surgery, duration of operation and position of patient on operation list and

-the in vitro disinfecting efficiency

 Materials and methods

The present study was conducted at Govt. medical college  hospital(GH), and a private multi specialty hospital  from October 2008 to June 2009. Two OTs ventilated without any special air filters (one for emergency -EOT & another for elective cases -MOT) from GH  and two OTs with modern facilities and  HEPA filters from a private multi specialty hospital (one for routine surgeries – PV I & another for cardiac and orthopedic  – PV II) were included. Ethical clearance was obtained from Institutional Ethical Committee.

After   fumigation with formalin on Sunday, sampling was done daily for 6 days from Monday (Day 1) to Saturday (Day 6). For another one week product X was sprayed daily and sampling was done everyday. Microbiological surveillance was done before the first surgery (morning -M) and at the end of the day (evening- E) by three different methods (settle plate, air sampler and surface swab). This was followed in all the four OTs daily.

Settle plate  :-Number of sheep  blood agar plates (10 cm size) as decided by the size of OT were exposed  at  different  areas  ( washing  room, OT table, OT corner, doctor’s room)  for  30mts  because  any  minor  surgery  takes  30mts-1 hr5 and     incubated at  37˚C  for  48hrs. If growth was observed, colony forming units (cfu) were counted followed by microscopy, biochemical and antibiotic sensitivity tests.

The acceptable limit of cfu was calculated by using the formula based on the colony count, area of the plate exposed, and the duration of exposure and it  is fixed as 10 instead of 12  to certify the OT is safe for surgery.6



Air sampler :-As recommended by the manufacturer, blood agar strips in Reuter’s centrifugal sampler , operating on  impaction principle7was used to collect air samples for 8 mts at different areas of OT (A/C, 6ft height and 1 feet height above the OT table) . They were incubated at 37˚C for 24 hrs and the cfu were counted followed by identification of bacteria. Colonies were counted based on the recommendations,

cfu /m3 = No. of colonies on agar strip×25 / Sampling time (in mts)

The acceptable upper limit is 180 cfu /m3 which approximately corresponds to 10 colonies settling on a plate.6

                                   Surface swab:-Swabs were collected from different points   (washing room, OT table, light, A/C, Boyles’ apparatus, drug rack) using peptone water and Robertson’s cooked meat medium (RCM). Peptone water swabs were streaked on nutrient agar, MacConkey and blood agar plates, incubated at 37˚C for 24hrs and the colonies were subjected to biochemical tests for identification.

Swabs collected in RCM were heat treated (80˚C-1 hr.) 8 and incubated at 37˚C for 10 days. Smears from these tubes were stained with Gram’s and examined for Clostridium tetani spores. Tubes showing spores were subjected to anaerobic culture on blood agar using Gaspak system   and aero tolerance test.9,10 Details of the results were recorded.


During the period of surveillance, the patients operated were followed for signs and symptoms of SSI. Any wound discharge was collected and processed for further analysis. Details of the patients and   risk factors for SSI were recorded if any.

To test the efficacy of product X one ml of 0.5% and 1 % disinfectant in peptone water   were prepared in aliquots. To this, 200 μl of stock culture of pathogen was added. Samples were plated after 10, 30 and 60 mts, incubated at 37˚C for 24 hrs and observed for any growth.  Aliquots of peptone water with distilled water mixed in the same proportion as disinfectant were plated to serve as controls. This simplified microbicidal activity test was done against common isolates like Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Enterobacter sp, Proteus vulgaris, Salmonella typhi, Pseudomonas and Acinetobacter sp.11

Statistical analysis was done by using variance ratio(F) for cfu of formalin & product X by settle plate as well air sampler, cfu for settle plate & air sampler, cfu for GH & private and cfu for morning and evening. Chi square test was also applied.


Since product X was sprayed on all days and formalin was fumigated on Day 6 only, to get an unbiased data, cfu on day 1 was considered very apt to compare the  two quantitative methods, settle  plate and air sampler . The cfu over the OT table was analyzed since it represents the real risk of SSI.

By settle plate after formalin fumigation in both OTs of  GH, M & E-cfu   on OTT exceeded. In PV I, both M and E -cfu were within limit. In PV II, the M -cfu  was  within limit, but E – cfu exceeded .  With product X in both OTs of GH, the M- cfu on OTT was within limit but E- cfu exceeded.  In PV I & II  ,both M and E- cfu were within limit.(Table1)The variances in cfu between formalin and product X were different( p < 0.01)

By air sampler after formalin, in all the four OTs (private and GH), both the M & E- cfu 1ft ht above the OTT, were within limit. Same finding was recorded with product X also.(Table1)The variances in cfu between formalin and product X were different (p < 0.01).By settle plate or air sampler, the cfu were less with product X compared to formalin, the M- cfu  were less than E- cfu  and the cfu in the private OTs were less compared to GH.(Table1)The variances in cfu between settle plate and air sampler were different (p <0.01).The variances in cfu between the morning and evening samples based on settle plate was different(p < 0.05), but there was  no statistical significant difference by air sampler. The variances in cfu between the OTs with and without special air flow arrangement was statistically significant by settle plate and air sampler(p < 0.01).

When the cfu was analyzed  from day 1 to 6,   no gradual decrease or increase was observed . On certain days this was either increased or decreased by settle plate as well by  air sampler after  formalin fumigation .Same results were observed with product X  also. (Table 2)

Organisms  isolated in GH by all the 3 methods:

                                    (settle plate ,air sampler and   swabs in   peptone water ) after formalin were Acinetobacter baumanii, CoNS, MSSA and micrococci . In private , aerobic spore forming bacilli and micrococci were isolated .Same isolates  were observed  respectively in GH and private with product X also.  Acinetobacter baumanii was sensitive to amikacin, ciprofloxacin, cefaperazone+sulbactam, ceftazidime, imipenem, meropenem, piperacillin +tazobactam , resistant to amoxicillin+clavulanic acid , and cefpirome. CoNS was sensitive to amikacin,ciprofloxacin, sparfloxacin, cefpirome, clindamycin, vancomycin , linezolid, cefoxitin and teicoplanin , resistant to amoxicillin+clavulanic acid, erythromycin and azithromycin. MSSA was sensitive to amikacin ,ciprofloxacin, sparfloxacin, cefpirome ,clindamycin, vancomycin, linezolid,teicoplanin, intermediate sensitive to azithromycin, resistant  to  amoxicillin + clavulanic acid , and erythromycin. RCM was negative for Clostridium tetani in both GH and private OTs.

Of the total 81 cases operated in GH, 11(14%) developed SSI.  In MOT, 2  out of 38 (5%)  and in EOT, 9 out of 43 (21%) developed SSI. There is no significant association between the nature of OT (EOT &MOT) and SSI(x2 =2.99). After formalin, 6 out of 35 (17%) and after product X, 5 out of 46(11%) developed SSI. (Table 3) No SSI was observed in private OTs. (Table2)The sterilizing agent has no association with the occurrence of SSI (p <0.05).


Of the 11 SSI cases, 5 were contaminated, 3  clean contaminated and 3 dirty cases. Duration of surgery in most of these cases were more than 60 mts. Of this 11 cases 9 were operated from EOT(Table3) Infection rate was NIL in clean cases.(Table 4) 4 out of 9 SSI cases  (dirty1, contaminated1, clean contaminated 2) were operated as first case in the EOT on that particular day. Of the 11 SSI cases, 8(73%)  were culture positive.    Escherichia coli was isolated from 6 cases(55%), ESBL producing Klebsiella pneumoniae from one case (9%) and  MSSA from one case (9%). Three(27%) pus cultures showed no growth aerobically.


                    Escherichia coli  was sensitive to ciprofloxacin, amikacin, amoxicillin+ clavulanic acid and pipercillin +tazobactam,intermediate sensitive to ceftriaxone, ceftazidime, resistant  to ampicillin and gentamicin.  ESBL producing Klebsiella peumoniae was sensitive to amikacin, and  intermediate sensitive to ciprofloxacin and gentamicin . MSSA was sensitive to amoxicillin + clavulanic acid , amikacin,sparfloxacin,cefpirome,clindamycin, vancomycin ,linezolid ,teicoplanin cefoxitin, intermediate sensitive to azithromycin. ciprofloxacin, resistant to  erythromycin.

SSI was observed  even  when the  cfu was  within limit by settle plate  after formalin. Same observation was made with product X. On the other hand   SSI was NIL when the cfu was above the limit. SSI was recorded even when the cfu by  air sampler were within limits.



The microbicidal activity of product X against Staphylococcus aureus ,Escherichia coli, Klebsiella pneumoniae, Citrobacter koseri, Enterobacter and  Salmonella  was  achieved in 10 mts with  0.5% and 1 % concentrations . For Proteus vulgaris, Pseudomonas and Acinetobacter baumannii it was observed at 30 mts with 0.5 % and  10 mts with  1 % concentration respectively.


The effective use of disinfectants and sterilization procedures in OT is critical for the prevention of post operative infections. A totally sterile environment is neither feasible nor advisable to direct all resources to achieve that. However, evaluation of cleaning, disinfection and sterilization methods is necessary for monitoring and to set policies.  “Fogging”, the nebulization of a disinfectant, especially formalin, is employed by most hospitals in India to decontaminate the OTs. However various objections have been raised against formalin.6






By  settle plate  after formalin in both the OTs of GH, even the M- cfu exceeded the limit. In PV I & II OTs, it was not only within  limit but also  much less than that  of GH. (Table1) This may be due to the meticulous cleaning before formalin fumigation and routine  adherence to strict theatre protocol in private hospital.12Sharma S et al13 suggested that formaldehyde is a high level disinfectant and its decontaminating value is undeniable and also agreed upon  that fumigation cannot replace manual cleaning and may in fact cause a false sense of security leading  to the abandonment of more effective infection control measures. Another reason for the low cfu in private OTs may be due to HEPA filters as observed by Peter G et al14 and Crimi P et al15



The E- cfu exceeded the limit  in both OTs of GH. In PVI it was within limit but in PVII ,it exceeded.(Table1) This discordance may be attributed to other   factors like human activity , number of persons , type  and  duration of  surgeries.16

By settle plate   after product X , in PVI & II, both  M- & E – cfu  were  within  limit.  In both OTs of GH,   M- cfu  was within limit  but E-cfu exceeded. (Table1)   This may be explained that disinfectant must be effective initially and its effect starts waning as the day progresses. If used in between the operation , it may be effective for longer duration.




Whether it was formalin or product X, the cfu within limit  or exceeded, the E-cfu was higher than M-cfu. (Table1) This may be due to the  human activity which significantly increases the microbial contamination and reaches the peak at the end of the day in  OTs and  was established by Geeta Mehta2, Ford CR et al17 Casewell MW et al18 Pasquarella C et al19 and  Ekhaise FO et al.20 The cfu after  product X was always less when compared to formalin indicating  its efficacy  is not less than that  of formalin .

By air sampler whether it is formalin or product X, in all four OTs, both M & E-cfu at 1 feet ht above the OTT, were within   limit.  Whether it is settle plate or air sampler, the M-cfu (compared to E-cfu), both M & E –cfu after product X  (compared to formalin), and cfu of private OTs (compared to GH) were less. Though the above findings correlated by both methods, the most important factor “the acceptable limit of cfu ” did not correlate, that is  by settle plate it exceeded whereas by air sampler the cfu was within limit for both sterilizing agents. This discordance is explained by the fact that   air sampler calculates suspended particles whereas settle plate calculates the settling large bacteria carrying particles.21These settling particles are more prone to cause SSI than the suspended ones.   Hence this economic and simple settle plate method has more practical application in reflecting the risk of infection.

Human activity and related factors have more influence on cfu , whether it is measured by settle plate or air sampler . It is evident that there is no gradual decrease or increase  of cfu from day 1- 6. (Table 2)



In this study, organisms isolated were same by all three methods. It may vary because each method detects different type of air particles. Surface swab is a qualitative method identifying the prevalent organisms and its sensitivity pattern. This would reveal more information about the seriousness of the contaminant.

RCM was negative for Clostridium tetani in both GH and private  OTs. Kelkar U et al6 did surveillance of OT where he isolated Clostridia only twice out of 276 evaluations and concluded that this could be a tool for good sentinel surveillance only. The age old traditions of detection of anaerobic spores of Clostridium tetani are losing ground with onset of more awareness on theatre sterilization. Routine testing for the anaerobes may not be essential except when there are suspected cases of Tetanus or Gas gangrene attributed to operation in a particular OT. But it may be ideal to survey the OTs for anaerobes when newly constructed, any remodeling or structural alterations are done. In such situations which will have trust worthy safety of the theatre.

In GH where there was no special air filters, the SSI rate was 14%. Previous  reports in India ranges from 4% – 30%.22 In GH, SSI after formalin and product X was 17% and 11% respectively indicating that  product X was more effective  than formalin. In EOT, SSI (21%) was higher than MOT (5%). (Table 3)  which may be due to the type of surgeries and host factors.

Whether settle plate or air sampler, after   formalin or product X, SSI was observed even when the cfu was low, on the contrary no SSI was noticed with high cfu.  Hence cfu and SSI did not correlate. This strengthens the fact that SSI is mainly due to host factors and it is of endogenous origin.


In this study, infection rate was nil in private hospital where there were special air flow arrangements like HEPA filters. Geeta Mehta,2 Dharan S et al23 also observed that in OT with HEPA filters there is less infection. Knobben BAS et al24 revealed that laminar flow system will decrease intra-operative contamination during total hip or knee replacements.

Among the total SSI, organisms isolated were,Escherichia coli 55%,  ESBL producing Klebsiella pneumoniae 9%and  MSSA 9%. No growth was observed aerobically in 27% of pus culture.  Nichols RL et al 25 also found that the normal endogenous microflora of the surgically resected organ were the most frequently isolated pathogens.

Two schools of thoughts were recorded about the origin of SSI, some advocate this as   exogenous but others established as endogenous. Many studies observed the role of exogenous and endogenous sources.26,27,28 The periodic screening and treatment of carriers will minimize the risk of transmission which was not assessed in this study. Many studies observed1,2,23,24 that wound infections can be controlled by length of operation. This study also revealed the same.  Some observed low sepsis rate in first operated cases1, 29 but in this study even first operated 4 cases were infected (dirty-1, contaminated-1.clean contaminated-2) (Table4) and the duration recorded was more than one hour. Hence infection rate depends on duration & type of surgery, operating team, endogenous and exogenous flora.



The microbicidal activity of product X against common isolates was good at 1% concentration within 10 mts. Hence, such products with proven efficacy may be used as an alternative to formalin. The environments in the OTs are dynamic and subject to continuous change. Good infrastructures do not mean a safe environment as human make a greater difference in making the environment unsafe.


Air sampler measures the microbial burden more  accurately. Settle plate is a direct indicator of SSI risk.  Clostridium tetani as an indicator of air quality may be limited to special situations only. Whereas organisms isolated from peptone water swabs   and their sensitivity pattern indicate the seriousness of the contaminant which   can guide the infection control programme effectively.  Frequency of surveillance depends on number & type of surgeries, out break of post operative infection and availability of resources. Routine surveillance for any OT may be suggested for every two months and for septic OT every month.  Newer less toxic disinfecting agents are alternative to formalin and  may be used at short intervals for better out come. The extra cost spent on this is worth considering the long term impact of formalin, an irritant, toxic, corrosive, and carcinogenic chemical. Special air flow arrangement plays a role in the maintenance of air quality. Correlation between microbial burden and SSI by any method is not absolute and it indicates only the source of post operative infection. Moreover fogging cannot replace manual cleaning. Since human activity plays a major role in microbial air quality, meticulous cleaning and strict adherence to OT protocol are essential. This miniature study may be done in an expanded manner   for   sufficient duration before a set of standard guidelines are established.



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Table 1: CFU on day 1 on OTT by settle plate and air sampler














Formalin Product  X Formalin Product X Formalin Product X Formalin Product X
Settle plate 13 18 7 11 16 57 9 14 1 4 0 2 0 14 0 9
Air sampler 84 118 28 138 72 163 19 94 06 22 0 22 0 22 0 19

*EOT-Emergency Operation Theatre      *OTT –OT Table

*MOT-Major Operation Theatre















Table 2: CFU from day 1 –  6 by settle plate & air sampler




DAY        Formalin Product X
  Settle Plate     Air sampler SSI   Settle  Plate     Air sampler SSI
         OTT 1footCFU/m3      OTT 1footCFU/m3


I 13 18 084 118 2  07    11    028 138 0
II 14 35 131 159 0 02 43 031 163 0
III 13 29 169 178 1 04 39 038 103 0
IV 16 27 153 166 2 09 41 019 059 1
V 17 25 131 159 0 03 64 013 131 2
VI 15 09 131 184 0 05 16 019 081 1




16 57 072 163 0 10 14 019 094 0
II 21 40 156 147 0 07 40 028 056 0
III 21 39 113 178 0 08 20 019 147 0
IV 20 54 088 169 1 11 24 053 041 1
V 21 37 131 188 0 12 28 020 125 0
VI 23 20 122 181 0 13 24 065 116 0
               PV1    1 01 04 006 022 0 00 02 000 022 0
II 03 05 044 047 0 02 43 022 078 0
III 05 09 038 066 0 01 02 003 028 0
IV 09 41 025 038 0 02 04 016 044 0
V 04 09 034 075 0 03 10 028 044 0
 VI 05 11 038 O66 0 04 19 013 013 0
PV2 I 00 14 000 022 0 00 09 000 019 0
II 09 19 022 034 0 03 15 003 091 0
III 06 32 038 066 0 01 02 000 028 0
IV 05 17 025 038 0 00 03 019 022 0
V 04 09 028 044 0 00 17 034 059 0
VI 05 11 038 066 0 02 42 019 081 0







































Table: 3 SSI rate after formalin and  product X  application






Formalin          Product X Total











No. of


Infected No. of


No % No %
MOT    15   1   7% 23 1 4% 38   2 5%
EOT 20 5 25% 23 4 17% 43 9 21%
Total 35 6 17% 46 5 11% 81 11 14%






Table: 4 Factors influencing SSI


of cases

Clean Clean


Conta Dirty
EOT (F) 5 0 0 2 3
EOT (X) 4   3 1  
MOT(F) 1 0 0 1 0
MOT(X) 1 0 0 1 0







1  Dr.S. Poongodi @ Lakshmi, M.D,

Professor of Microbiology, TiruneveliMedicalCollege,Tamilnadu

2  Dr.N. Palaniappan, M.D,

Professor of Microbiology,TiruneveliMedicalCollege,Tamilnadu

3 Dr,M. Kannan, M.D,D.A,

Professor  of Anaesthesia ,  TiruneveliMedicalCollegeHospital, Tamilnadu

4 Dr.S.Nithya Gomatheeswari, M.D

Asst.Professor  of  Microbiology, Chengleput

Corresponding Author:

Name   : Dr. S. Poongodi @ Lakshmi,M.D,

Address: Professor of Microbiology,

TirunelveliMedicalCollege,Tirunelveli ,Tamilnadu. -627011

Phone numbers: 9597720438   E-mail address – kodi68@gmail.com [2]