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 ~  Abstract
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgement
 ~  References

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Year : 2003  |  Volume : 21  |  Issue : 4  |  Page : 265-267

Treatment of discarded blood units: Disinfection with hypochlorite / formalin versus steam sterilization

Department of Microbiology, Choithram Hospital and Research Centre, Indore - 452 001, India

Correspondence Address:
Department of Microbiology, Choithram Hospital and Research Centre, Indore - 452 001, India

 ~ Abstract 

Blood bank regulations and bio medical waste rules of India advocate disinfection of contaminated blood units. Incineration is not recommended due to poly-vinyl chloride (PVC) content of blood bags. This study was designed to evaluate the efficacy of chemical disinfection of blood units deliberately contaminated with Staphylococcus aureus and E. coli with 1 and 6 % hypochlorite, 10% formalin and 33% formaldehyde and autoclaving of blood units contaminated with the above mentioned vegetative forms and B. stearothermophilus spores. Only 33 % formaldehyde could bring about 5 log reduction of bacteria but it is highly irritating and toxic. Autoclaving at 15 lbs pressure for 2 hours uniformly inactivated the vegetative forms and B. stearothermophilus spores. Thus, autoclaving of PVC blood bags is a safer and reliable method compared to chemical disinfection.

How to cite this article:
Chitnis V, Chitnis S, Patil S, Chitnis D. Treatment of discarded blood units: Disinfection with hypochlorite / formalin versus steam sterilization. Indian J Med Microbiol 2003;21:265-7

How to cite this URL:
Chitnis V, Chitnis S, Patil S, Chitnis D. Treatment of discarded blood units: Disinfection with hypochlorite / formalin versus steam sterilization. Indian J Med Microbiol [serial online] 2003 [cited 2020 Nov 28];21:265-7. Available from:

Six lakh units of blood are collected annually in India.[1] Possibly 40,000 - 60,000 units need decontamination being positive for HIV, Hepatitis B, Hepatitis C, suspected bacterial contamination or expiry (figures based on 8 - 10 % annual discarding of blood units at our institute). As per Indian Bio medical waste (BMW) rules 1998,[2] these units are required to be chemically disinfected or autoclaved prior to disposal and disinfection is often done by hypochlorite or formalin treatment. Poly vinyl chloride (PVC) content of blood bags discourages incineration. Scientific data to support autoclaving or chemical disinfection for blood units is lacking. Hence, a study was conducted to compare autoclaving and chemical disinfection by hypochlorite or formalin for PVC blood units that were deliberately contaminated with bacteria / bacterial spores.

 ~ Materials and Methods Top

Hypochlorite (6%; Microkill, Bhopal, India), 1% hypochlorite, formaldehyde (33% SDS labs, India) and 10% formalin solution were used for testing.
The neutralizer solution contained 3 gm lecithin (Sigma chemicals), 30 mL polysorbate 80 (Sigma), 5 g sodium thiosulphate (Analar), L histidine (Sigma), 20 mL potassium dihydrogen phosphate pH 7.2 (Analar) and 990 mL of purified water.
Blood bags
PVC blood bags (Terumo Pen Pol, India) expired or with transfusion reaction from the blood bank at Choithram Hospital and Research Centre (CHRC), Indore were used.
A horizontal autoclave with a thermograph and digital temperature display dedicated for waste in Microbiology department at CHRC was used.
Challenge organisms
Multiple clinical isolates of Staphylococcus aureus and  Escherichia More Details coli from Microbiology department at CHRC and Bacillus stearothermophilus strain from Haffkine Institute, Mumbai, were used for inoculum preparation. Fresh overnight bacterial growth on nutrient agar slants was harvested in sterile normal saline. The turbidity was adjusted to 0.5 McFarland standard (1 x 108) CFU/mL. Sporulated cultures on nutrient agar slants incubated at 550C for 5 days were used for making challenge suspensions of B. stearothermophilus.
Chemical disinfection study
Ten millilitre bacterial suspension was introduced through a tubing of the bag using sterile syringe and needle and the tube was resealed. The blood mixed with the bacterial suspension and 30 mL disinfectant was added by syringe. The tube was resealed and mixed for 30 minutes. After 30 minutes exposure, 5 mL blood was withdrawn and added to 50 mL neutralizer. Dilutions in sterile normal saline were subjected to total viable count using nutrient agar plates. Each experiment included both test organisms with 10 blood units; 1 control, 4 test blood units for exposure with 1% and 6% hypochlorite, 10% formalin and 33% formaldehyde for each organism. The experiment was repeated two times. Thus 30 bags, 6 for control and 24 test bags were included in the study.
Autoclave study (vegetative forms)
Ten millilitre of bacterial suspension was added to PVC bag as described above. The blood bag was exposed for one sterilization cycle (15 pounds for 2 hours). After autoclaving the blood bag was cut open aseptically in bio-safety hood and one cubic centimetre of coagulated blood each from four corners and centre were removed with sterile scalpel and crushed in sterile pestle and mortar. Crushed material was added to 50 mL of sterile normal saline before subjecting to viable counts as above. A total of 40 blood bags, 10 bags for test and 10 non autoclaved bags as control per organism were included in the study.
Autoclave study (spore)
Preliminary study with B. stearothermophilus spores inoculated in blood showed 2 to 3 log reduction even at room temperature and hence the experiment was modified. The spore suspension (2 mL) in sterile 5 mL screw capped glass vial was inserted aseptically by making a 2 cm. slit in the top portion of the bag. The bag was sealed with a hot rod. After autoclaving, the vial was removed from the coagulated blood and suspension subjected to viable count study after dilutions. The plates were incubated with 95% humidity at 550C for 5 days. Control tubes of spore suspension kept at room temperature for 3 hours were also subjected to viable count study. A total of 10 blood units were used for each of the 10 autoclave cycles.

 ~ Results Top

Survival of challenge bacteria in blood bags is as shown in the table. Hypochlorite (1,6%) and 10% formalin exposure could bring about only 2 to 3 log reduction in the bacterial challenges. Only 33% formaldehyde, could satisfy the criteria of 5 log reduction in bacterial counts. Autoclaving resulted in the sterilization of S. aureus, E. coli and the spores of B. stearothermophilus.

 ~ Discussion Top

The treatment of PVC blood units forms an integral part of BMW management. Chemical disinfection with hypochlorite and formalin is being practiced since incineration of PVC releases dioxins and furans.[3] Disinfection with hypochlorite is economical but albumin, serum, plasma and blood inactivates chlorine.[4] Our earlier study has documented the inefficiency of 1% hypochlorite in decontaminating blood containing hypodermic needles.[5] The disinfectant used in our study was limited to 30 mL as the PVC blood bag is already filled with 400 mL blood and 65 mL of citrate phosphate dextrose adenine (CPDA) solution (anticoagulant). The addition of excess disinfectant seems impractical and the present findings strongly question the recommendation of hypochlorite and 10% formalin use for disinfection. Although 33% formaldehyde effectively disinfected the blood bags, it is very toxic and even addition of 30 mL formaldehyde was difficult because of irritating fumes coming out during and after addition.
The objection for autoclaving sealed PVC bags is said to be penetration of steam. The liquids in sealed containers can be sterilized by steam but blood is coagulated by heat and therefore adequate steam may not be generated in the sealed bag. Further, the temperature achieved in the plastic bag kept in autoclave has been reported to be lower than the achieved temperature.[6] Hence, the time of exposure in autoclave was increased to 2 hours (15 pounds or 121C). Moreover, the microbiology waste contains massive bacterial loads hence the routine practice at our institute is to run a two hour autoclave cycle. The study did not include viruses like HIV, HBV and HCV but the fact that B.stearothermophilus spores (universal biological indicator) were inactivated confirmed efficacy of sterilization.
To conclude, the BMW authorities should discourage the chemical disinfection of PVC blood units and autoclaving should be uniformly recommended. It is also suggested that the blood banks in the hospitals could pass on the blood units to microbiology laboratory autoclave for treatment.

 ~ Acknowledgement Top

The present study was supported by Council of Scientific and Industrial Research, India.  

 ~ References Top

1.Mohanta GP, Manna PK, Manoj MD. Plasticised PVC as blood bags. The Indian J Hosp Pharmacy 1999;1:14-17.  Back to cited text no. 1    
2.Bio - medical waste (Management and Handling) Rules, 1998: Ministry of Environment and Forests Notification, New Delhi;1998.  Back to cited text no. 2    
3.Travis C, Hattemer Frey HA. Human exposure to dioxin. Sci Total Environ 1991;104(1-2):97-127.  Back to cited text no. 3    
4.Rutala WA, Weber DJ. Uses Of Inorganic Hypochlorite (Bleach) In Health Care Facilities. Clinical Microbiological Reviews 1997;10:597-610.  Back to cited text no. 4    
5.Chitnis V, Chitnis S, Patil S, Chitnis DS. 1% Hypochlorite is inefficient in decontaminating blood containing hypodermic needles. Indian J Med Microbiol 2002;20(4):215-218.  Back to cited text no. 5    
6.Lauer JL, Battles DR, Vesley D. Decontaminating infectious laboratory waste by autoclaving. Appl Env Microbiol 1982;44(3):690-694.  Back to cited text no. 6    
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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

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