SETTLE PLATE CULTURE TECHNIQUE FOR OT AIR SAMPLE

 

Purpose:

The objective of microbial surveillance test is

To detect if there is any bacterial colonization of indoor air of OTs even after cleaning and fumigation.

To estimate the bacterial colony-forming unit (CFU) rate of indoor air and to identify the type of bacterial contamination of OTs.

Principle:

Medical science has evolved tremendously since the time of Louis Pasteur who gave the germ theory of disease. Since then, the prime responsibility of the physician has become the prevention and control of various microbes at every place, especially in health care facilities. Hospital-acquired infections are a major cause of morbidity and mortality in the patients coming to hospitals for various reasons. The environment of operation theatre (OT) plays a major role in the postoperative recovery of the patients. Infection acquired in OTs leads to increased morbidity and mortality, prolonged length of hospital stay, increased expenditure of patient and hospital.  Using good theatre practice, standard cleaning, and proper sterilization, infections in the OTs can be minimized.

Equipment and reagents:

    Equipment: Sterile plates, laminar air flow, Incubator

   Reagents/ Media: Blood agar, Sabouraud Dextrose agar, Stains, Biochemical reagents

Sample Collection

    The sampling method and the number of samples collected will be influenced by the potential contamination circumstances.

Pre Sampling Inspection

Several preliminary concerns must be addressed when designing a microbiologic environmental sampling strategy, which should meet with the below four Circumstances of environmental sampling namely

To support an investigation of an outbreak of disease or infections when environmental reservoirs or fomites are implicated epidemiologically in disease transmission

Limit microbiologic sampling for quality assurance purposes

To monitor a potentially hazardous environmental condition, confirm the presence of a hazardous chemical or biological agent, and validate the successful abatement of the hazard

Pre sampling inspection should be performed prior to sampling to ensure that area is well prepared for sampling. This is usually performed by the technician of the sampling area in cooperation with hospital staff and supervised by Infection control personnel.

The inspector should evaluate and assure that these criteria’s are met prior to each sampling event, to identify the conditions that may affect or interfere with the proposed testing:

All new or reestablishment work is completed.

The ventilation system had been running continuously for at least 24 hours following the completion of structural work, and cleaning.

 All engineering commissioning procedures has been completed.

All fixed and portable equipments placed in their places.

Cleaning of Floors, all surfaces and equipment are finished 24 hrs prior to the investigation by 24 hours. A Checklist for Monitoring Cleaning Prior to Sampling (Appendix B) should be used by the head nurse of the sampling area to ensure proper cleaning.

Ducting and air diffusion sites (inlet, outlet) are cleaned prior to sampling.

Minimizing indoor traffic at all times when possible.

The sampling personnel should be completely familiar with the sampling protocol and type of the particular method to be used in different situations.

In case of microbiological out breaks by one or more microorganisms. The following should be considered:

The decision to sample should be based on the extent and location of any suspected contamination and the potential for the contaminant to migrate and the activities for which the facility is used.

The decision to collect environmental samples should be made by the infection control committee.

All personnel who enter the contaminated area must follow the safety and infection control plan developed for that particular site.

Shutting down the ventilation system serving the contaminated area may be necessary only in special cases to avoid dispersing certain contaminate (i.e. Mucor species, Bacillus anthracis etc).

Settle Plate Method

Procedure:

Media-containing plates are exposed to the atmosphere face-up to collect gravity-settling particles. For bacterial sampling, SBA and MCA were used, while for fungal sampling, Sabouraud Dextrose agar (SDA) was used. 

Media plates transported to surveillance sites are labeled with sample number and date of sample collection.  The plates must be kept open at a height of 1 m above the ground and 1 m from the walls and exposed for 1h (ie) 1/1/1.

The bacterial sampling Petri plates are aerobically incubated for 24 to 48h at 37°C, while the fungal plates are incubated for 7 days at 25 to 27°C in the BOD incubator.  

These plates are observed for the presence of growth after incubation and the number of colonies per plate is counted.   Bacterial culture reports will be reported after 24-48 hrs with colony forming units (CFU).

The mean number of viable bacteria or fungi by cubic meter (m3) of air can be calculated and the results will be written as colony forming unit per cubic meter of air (CFU/ m³). 

Calculation:

In addition, Omeliansky's formula colony forming unit (CFU) per cubic meter of air (CFU/m3) is calculated.

N = 5a × 104 (bt)−1

Where, N = colony forming unit per cubic meter of air (CFU/m3)

a = number of colony forming unit per Petri plate (CFU)

b = surface area of Petri plate (cm2)

t = time exposure (min).

Final identification is done by following standard bacteriological techniques, and fungal identification is done by Lactophenol Cotton Blue (LCB) mount.

Interpretation

Settle Plate:

All isolates were categorized into 3 broad categories

1.   Normal flora: e.g. Coagulase-negative Staphylococci (CoNS),

2.   Contaminant: e.g Bacillus spp.,

3. Pathogen: e.g. Klebsiella spp., Staphylococcus aureus and Pseudomonas aeruginosa.

In OTs and Labour Room, even a single colony of Staphylococcus aureus and fungus is considered unsatisfactory. Repeat the cleaning procedure

Biological reference intervals or clinical decision values:

An empty theatre bio load should not exceed 35 CFU/m3.

During surgery bio load should not exceed 180 CFU/m3.

Area	Hazard / Indicator	Result	Interpretation	Action
ICU	Total bacterial	Counts < 50 CFU/m ³	Satisfactory	N/A
		Counts > 50 cfu/m ³	Unsatisfactory	Take ICU out of use, clean thoroughly and re-test
	Aspergillus sp.	Counts < 5 cfu/m ³	Satisfactory	N/A
		Counts > 5 cfu/m ³	Unsatisfactory	Take ICU out of use, clean thoroughly and re-test
Protective environment room (PE)	Total bacterial	Counts < 35 cfu/m ³	Satisfactory	N/A
		Counts > 35 cfu/m ³	Unsatisfactory	Take room out of use, clean thoroughly and re-test
	Aspergillus sp.	No single  Aspergillus sp.	Satisfactory	N/A
		Counts > 0.1 cfu/m ³	Unsatisfactory	Take room out of use, clean thoroughly and re-test
Conventionally (empty) ventilated theatre	Total bacterial	Counts < 35 cfu/m ³	Satisfactory	N/A
		Counts > 35 cfu/m ³	Unsatisfactory	Take theatre out of use, clean thoroughly and re-test
	Aspergillus sp.	No single Aspergillus sp.	Satisfactory	N/A
		Counts > 0.1 cfu/m ³	Unsatisfactory	Take theatre out of use, clean thoroughly and re-test
Ultra-clean theatre (air leaving final diffuser)	Total bacterial	≤0.5 cfu/m3	Satisfactory	N/A
		Counts > 0.5 cfu/m ³	Unsatisfactory	Take theatre out of use, clean thoroughly and re-test
	Aspergillus sp.	No single Aspergillus sp.	Satisfactory	N/A
		Counts > 0.1 cfu/m ³	Unsatisfactory	Take theatre out of use, clean thoroughly and re-test

       Reference:

1.Guidelines for Environmental Infection Control in Healthcare Facilities.  Recommendations of CDC and the Healthcare Infection Control Practices Advisory Committee (HICPAC). 2003 (updated 2019) Pg 103 – 110.

2.Myer’s and Koshi’s., Manual of diagnostic procedures in medical microbiology and immunology/serology – CMC, Vellore -2001(page: 101).