Biosafety In The Laboratory: Prudent Practices for the Handling and Disposal of Infectious Materials.


In 1984, the Centers for Disease Control (CDC) and the National Institutes of Health (NIH) jointly published a set of guidelines for the safe handling of pathogenic microorganisms[ ]. These guidelines, developed over a period of several years in consultation with experts in the field, remain the best judgments available; they are reproduced here in their entirety, as Appendix A. The reader should consult these guidelines in deciding on the appropriate level of precaution to use in the handling of a particular organism.

Guidelines for handling agents identified after the CDC/NIH publication are published as Agent Summary Statements in Morbidity and Mortality Weekly Report (MMWR), issued by the CDC. The Agent Summary Statement for human immunodeficiency virus (HIV)[ ]is reprinted here as Appendix B, and additional MMWR articles on HIV (“Recommendations for Prevention of HIV Transmission in Health-Care Settings”[ ][ ]) are reprinted here as Appendix C.

Throughout this and the following chapters, frequent reference is made to Biosafety Levels 1 through 4. These levels are described in the CDC/NIH publication (Appendix A). Table A.1 of this appendix summarizes the practices, techniques, and safety equipment prescribed for each level.


The risk of acquiring an infection in the laboratory is influenced by many variables. Among these factors are the health and immune status of the laboratory worker, the suitability of the laboratory for work with highly pathogenic agents, the characteristics and the concentrations of the microbe being handled, and the specific manipulations involved in its handling.

Studies of infections acquired by personnel working in microbiological laboratories have been carried out by several investigators over the past half-century[ ][ ][ ][ ][ ][ ]and have identified a number of potential human pathogens that are clearly more frequent causes of laboratory-acquired illnesses than are others. Organisms falling in this category are to be found among viruses, bacteria, rickettsiae, and fungi. Awareness of those species with a high potential for invading normal humans should lead to the use of appropriate precautions to minimize the risk of infection.

Among the agents that have been identified in recent years as posing the greatest risk of infection to laboratory and ancillary personnel of diagnostic laboratories are the virus of hepatitis B, Mycobacterium tuberculosis, and Shigella spp. [ ][ ][ ]. A partial list of other agents known to pose greater than average risk to laboratory workers includes Brucella spp., Salmonella spp., leptospires, Coxiella burnetii, Rickettsia spp., and Coccidioides immitis. The recently identified virus of AIDS (HIV), on the other hand, poses a low risk of occupational infection to laboratory workers, except to those working with concentrated virus suspensions[ ][ ]. The supplement to the CDC/NIH guidelines recommends, therefore, that HIVs be handled according to the standards and special practices of Biosafety Level 2 or 3, depending on the concentration or quantity of virus or the type of laboratory procedure used (see Appendix B).

No agent that is a component of the normal or abnormal microbial flora of man should be regarded as lacking totally in pathogenic potential, and all microorganisms should be handled with appropriate techniques. With the increase in research in virology in the past half-century, laboratory infections with viruses have increased relative to those caused by bacteria and mycoplasmas.

An important defense against infection with some viral agents is immunity induced by vaccination. Whenever a vaccine is available (see Table 5.2), its use should considered for those at risk of exposure prior to their handling of the virus in question. Under certain circumstances, when work with highly virulent agents is contemplated, it may be necessary to consider the administration of an experimental vaccine. Because of the potential risk of injury to the fetus from apparent or inapparent viral infection, special precautions, including temporary reassignment, may be considered for female personnel who are pregnant or are contemplating pregnancy.

All personnel working with infectious agents should have documented evidence of immunization with the vaccines required by most jurisdictions for admission to elementary school, e.g., diphtheria, tetanus, pertussis, poliomyelitis, measles, mumps, and rubella. In addition, vaccines for preventing infections with other agents to which they may be exposed, if available, should be offered, and in certain circumstances consideration should be given to making such immunization mandatory.

Acceptance of immunization against, or demonstration of proven immunity to, hepatitis B virus should be a precondition for the employment of all workers who will be handling human blood or body fluids. If the medical program of the hiring organization includes a serum bank, a sample should be obtained at the time of employment and stored in the frozen state, to provide a baseline for subsequent immunologic assays as required.


Microbial population constantly inhabit community and hospital environment. The hospital surfaces are often contaminated with flora excreted by patients, visitors and healthcare workers. The contaminated environmental surfaces are potential reservoirs for spread of microbial agents in hospital as well as community. Persistence of pathogens in hospital environment increases the risk of infection among susceptible host. Microbial population and colonization rate vary with different hospitals of different countries. Study of bacteriological profile of common sites of hand contact would help to locate the possible reservoirs of bacteria and to apply suitable disinfection techniques.

The bacteriological profile of frequently used objects (Non medical devices) yielded wide variety of organisms ranging from normal flora to potential pathogens like S. aureusAcinetobacter species, E coli and Pseudomonas species. The biometric attendance devices are widely used in hospital as well as community. Microbial colonization of such devices may spread the potential pathogens among unsuspecting users. In our study, 33.3% (8/24) specimens collected from biometric attendance devices showed growth of S. aureus of which 25% (2/8) were MRSA. Escherichia coli is commonly associated with community infections, isolation of ESBL producing E coli from these sites indicates possible role in serious nosocomial infections. Acinetobacter species and Pseudomonas species are well known nosocomial pathogens and their presence on biometric devices can lead to spread in the hospital and community. Similar studies have shown the presence of Acinetobacter species, Pseudomonas species and E coli on other inanimate objects of hospital. Manipal Teaching hospital has more than six hundred healthcare workers who mark attendance twice a day with these devices. Colonization of such devices by potential pathogens like MRSA and ESBL producing Gram negative bacteria indicates the possible spread of these pathogens among the hospital as well as in community population. Most of the healthcare workers before starting their duty and at the end of duty hours mark attendance with biometric devices. At the end of duty hours, healthcare workers wash their hands in their respective departments and then mark attendance on biometric devices. Their fingers may get contaminated with pathogens persisting on the devices. Healthcare workers rarely wash hands after use of biometric attendance devices. This may lead to dissemination of pathogens from hospital into community. Nancy S et al also reported 33.3% S. aureus isolation and 70% MRSA prevalence on biometric device surfaces.

Hospital elevator buttons are another frequently touched objects by healthcare workers, patients and visitors. Out of 48 specimens collected from elevator buttons, 22.9% (11/48) showed growth of S. aureus with 36.3% (4/11) MRSA. Sayeed et al reported 75% contamination of elevator buttons with S. aureus which is higher than our findings. Other potential pathogens were Pseudomonas species, Acinetobacter species and E coli. Kandel et al, reported isolation of Staphylococcus species, Pseudomonas species, coliform bacteria from elevator buttons. Healthcare workers including doctors and nurses frequently use elevators. This increases the risk of transmission of these potential pathogens to the patients. Similarly, finger contamination of medical students, visitors and staff by elevator buttons may spread these pathogens in the community as well. Elevator buttons are one of the neglected sites in a hospital, often not cleaned or disinfected and can become potential site for bacterial colonization.

Door handle contamination by potential pathogens has been recorded from the medical ward, surgical ward, ICU and post-operative ward. In a study by Odigie et al., S. aureusPseudomonas species and E coli were reported as common isolates from door handles. In a recent study, E coli was reported second most common bacterial isolate from door handles. Isolation of E coli from door handles of pediatric units could be threat for serious infections among neonates. In a similar study by Oie S et al, door handle contamination by S. aureus in a University hospital in Japan was 27% which is comparable with our study (16.2%). Saba et al, reported S. aureus colonization rate of 39% (47/120) from door handles, staircase railings and other point of contact in Teaching Hospitals. The MRSA colonization on door handles in our study was 30.7% (4/13) which is higher as compared to above study (17%). The risk of transmission of pathogens is more as nursing staff, clinicians and visitors frequently touch door handles during visit to wards.

Hospital telephone sets can be important source of nosocomial pathogens in various units. Contamination is likely to occur due to regular hand and ear contact of healthcare workers with almost no attempt at cleaning/disinfection. The contamination of telephone sets by potential pathogens was observed from Surgery, ICU, Pediatric, Gyanecology and Medicine wards. Culture of telephone sets yielded large number of Acinetobacter species. Isolation of multidrug resistant Acinetobacter species and E coli from ICU is alarming. S. aureus colonization rate of telephone sets was 20% (6/30) with 33.3% (2/6) MRSA. In a similar study by Zubair et al., lower, 9% (4/44) S. aureus contamination was reported. Bacterial contamination of other frequently touched sites like staircase railings pose the risk of transmission especially among the children who fondle railings during hospital visit. Similarly, contamination of water taps (wash room and drinking water) by nosocomial pathogens may lead to contamination of drinking water resulting into gastrointestinal disorders. Presence of potential pathogens on hand operated water taps of washroom increases the possibility of recontamination of hands negating the benefits of hand washing. Above mentioned sites were selected for this study due to frequent and unavoidable contact with these surfaces. These sites are very infrequently cleaned or decontaminated. In most of the teaching hospitals, routine cleaning involves hospital floor, working bench, table tops, nursing stations, dressing trolley etc. However, there is almost no practice of cleaning/disinfecting sites highlighted in this study. High rate of bacterial contamination in the above mentioned sites reflects poor hand hygiene among the healthcare workers and visitors as transmission occurs mainly through contaminated fingers.

Staphylococcus aureus is well known nosocomial pathogen with its ability to survive in hospital environment for several days. The ability of S. aureus and MRSA to form biofilm on inanimate objects prolongs their survival and spread. Ankit et al. from Nepal, reported 65.7% clinical isolates of MRSA from pus/wound swabs as biofilm producers, however data related to environmental samples is not available. Majority of MRSA isolates 62.5% (10/16) in our study were biofilm producers which is comparable with above mentioned report. This is alarming as MRSA isolates embedded in biofilm can survive long duration and can become potential source of MRSA associated nosocomial and community infections. Such infections are difficult to treat due to in vivo biofilm formation. Identification of the more frequently contaminated sites and the most commonly identified potential pathogen is important for infection control practices and promotion of new interventions.

Study of bacterial contamination of multiple sites of frequent hand contact in a hospital is the strength of our study. Biofilm formation by the most common potential pathogen S. aureus is important determinant for their survival and transmission.

This study has some limitations. The molecular characterization of the potential pathogens was not performed. We could not prove the association of pathogens isolated from objects and prevalent nosocomial infections. Biofilm property was studied only for S. aureus isolates. The study was conducted in one of the tertiary care hospital and results of the study may not be generalized.

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