Monitoring Medical/Biowaste Bags With Spore Strips as Biological Indicators and ‘False’ Negatives

It has been noted that a fairly large number of facilities use spore strips as their biological indicator for the monitoring of biowaste bags to be steam sterilized. These waste bags include regulated medical waste (RMW) treatment, biological waste and a variety of lab, clinic or media prep waste bags. The waste contents could be made up of various discarded agar slants, petri dishes, tubes of TSB or other such common lab waste that is likely to contain various strains of bacterial cells, including spore-forming gram-negative bacillus, E. coli, fungi, mold and/or yeast cultures. Isolate and contaminant cultures, body fluid wipes and bloody swabs and gauze can also be included with RMW and thrown into the biobags. Waste accumulates from the intensive care units (ICUs), pathology labs and surgical suites. The number and types of possible pathogens is very large. Included in this potpourri may also be antibiotic discard, cleaning tissues or cloths used to sanitize surface areas, or clean laminar-flow hoods that contain leftover sporicidal or antimicrobial spray, alcohols or disinfectants that may have been used in cleaning processes.

The waste bags usually contain an abundance of petri dishes, etc. that will liquefy under sterilization conditions. Agar slants, tubes of TSB or other growth media and numerous items containing collected or cultured contaminants will soon be free-flowing within the bag as temperatures reach the agar melting point and plates and tubes open up under pressure and temperature.

Other contributing medical/bio sites would include environmental/water-testing labs, tattoo facilities, dental offices, veterinary clinics and a huge number of public health labs. All of the aforementioned facilities are accumulating biowaste and it must be sterilized prior to disposal. Most of these waste bags sit in collection areas until they are sent off to be autoclaved, either on-site or off-site. Many sites use biological indicators (BIs) in the form of spore strips to measure the cycle’s effectiveness. Could just the presence of this hodgepodge of waste-bag items discarded into the bags have a localized affect upon the spore strips being used to monitor a sterilization cycle?

Prior to providing information pertaining to the question above, let’s look at why spore strips are being used as BIs for waste-bag cycles. The most common form of BI used as an indicator for monitoring sterilization cycles involving steam is the spore strip. Spore strips are fairly easy to use, they are the least expensive BIs to purchase and most individuals using BIs are familiar with the use of spore strips. Of the regulatory standards that require the use of BIs for monitoring medical or biological waste, they specifically mention and allow for the use of spore strips in autoclaves. When one is preparing for environmental laboratory accreditation, autoclaves must be monitored with spore strips or suspensions. Autoclave loads would also encompass lab/biowaste loads.1 Numerous agencies, including many state department of health agencies, follow the four proposed sterility assurance levels for microbial inactivation set by the U.S. State and Territorial Association on Alternative Treatment Technologies (STAATT) for the treatment of medical/biowaste. RMW/biowaste requires a Level 3 minimum for treatment. With Level 3, the use of a minimum challenge of log-4 population is used.

As an example of citing spore strip use, the recently approved Code of Mass. Regulations for the ‘Minimum Requirements For the Management of Medical or Biological Waste (State Sanitary Code Chapter VIII), section 480.150, E 4, states, “Testing shall consist of spore strips or a retrievable medium approved by the Department, which contain a 1.0 X 104 minimum challenge population of a bacterial indicator organism that is most resistant to any aspect of the treatment technology as outlined in the most recent medical waste treatment technology guidelines established by the STAATT or its successor, the International Society of Analytical Analysis of Treatment Technologies (IStAATT).” When first viewing this code, one would quickly see “spore strips or a retrievable alternative medium approved by the Department.” An easy choice for compliance would just be to use the spore strips with a population of log-4 and avoid getting approval by the Department for an alternative medium. Thus in a majority of cases, spore strips are used and at a population of log-4.

In order to test if a possibility exists where just due to the presence of the disinfectant or bactericidal cloths or wipes put into a waste bag and in close proximity to the spore strips could possibly have an affect upon the spore strips (at a population level of log-4) in such a way as to provide the user with a false negative, the following test was conducted.2 Three red bag autoclave waste bags were prepared. Each bag contained approximately the same volume of discarded petri dishes, lab paper towels used for cleaning flow hoods, discarded tubes of growth media and TSB, along with the usual lab discard of pipette tips and lab test discard. Into all three bags were placed spore strips with a population of log-4, four test strips per bag. Included in bags 1 and 2 were placed the moist paper wipes or paper towels that were used to clean flow hoods using bactericidal and disinfectant sprays. The spore strips containing spores of G. stearothermophilus and towels were intentionally placed near the top area of each bag. In this manner, steam penetration into the lower parts of the bag was not to be considered an issue. In bag 3, no towels containing traces of bactericidal spray were used. All three bags were run in the same cycle, 121 degrees Celsius exposure temperature with a prevac phase used. The exposure time was set at 4 minutes at 121 degrees C. This time was chosen as an “all survive” time for the spore strips under normal 121 degree C conditions. With this exposure time, all spore strips should survive, since the short exposure would be sub-lethal. The 4-minute time was elected from the spore strip certificate of analysis where the population was at 2.5 X 104 and had a resistance of 1.9 minutes. Using USP survive/kill calculation (log of population minus 2 times the D-Value) we had a ‘survive time’ of 4.5 minutes exposure to 121C where all the spore strips would survive the exposure.3 In our brief trial, we chose an even shorter time for exposure and used 4 minutes. Following an exposure cycle of 4 minutes at 121C, all spore strips were removed from the waste bags and the strips were aseptically transferred to tubes of TSB and incubated at 55 degrees C to 60 degrees C. Growth and no growth results are listed below after seven days of incubation.

With an exposure time of 4 minutes, the spore strips should have survived the cycle in all three waste bags. Even though all three waste bags were exposed to the same sub-lethal exposure time of 4 minutes at 121 degrees C, the spore strips in waste bags #1 and #2 did not show signs of growth while waste bag #3 without the tissues did grow and was not killed by the short exposure cycle. It was not the exposure cycle that killed the spore strips in bags #1 and #2 but the waste bag contents contributed to lethality.

Admittedly, additional tests should be run with various exposure times used and strip placement variation within the bags. However, I feel that this brief investigation allows for the ‘possibility’ that if one were using spore strips for monitoring waste bag sterilization, a false negative is within reason as a possible risk. A possible risk that even if ever so slight, simply should not be allowed to exist.

With typical waste sterilization cycles dealing with PreVac, exposure time and temperatures, most likely even a waste bag bioburden of 1012 or 1014 pathogens would likely be killed due to low moist heat resistance. However, if we are monitoring a cycle’s effectiveness with spore strips and a possibility exists where test results could be false, our confidence level for sterility assurance has been greatly lowered.

Over the past 15 years I have come into contact with numerous waste bag steam cycles where spore lethality just did not occur. Some were due to inadequate exposure time but by far a majority was due to inadequate air removal and thus poor steam penetration. Some cycles just did not include a PreVac phase and thus steam penetration was extremely difficult or impossible to obtain and some were mechanical vacuum failures. Time and temperature recordings were fine but the prevac was missing and TCs were measuring dry heat rather than moist heat within a bags content. ‘IF’ spore strips were used and ‘IF’ they were in contact with bactericidal or disinfectants in a very localized area in such a situation where adequate steam penetration did not occur and the strip should have survived, we could possibly have a failed cycle yet the spore strips would indicate adequate cycle parameters were met and a failed cycle not detected. The accompanying photo would show what an extreme biological indicator compromise situation could look like if the strips were allowed to come in contact with waste bag contents.

The spore strips could very likely be compromised and not provide valid testing or monitoring results. Even extreme, moisture saturated spore strips at a cycles end should be suspect of being compromised. Spore strips are not manufactured for or intended to be placed within liquid loads. Using them in such is contrary to ‘intended use.’

If a biological indicator is to be used in liquid or high moisture accumulation loads, it should be completely sealed from the possible affects that the load contents could have upon the indicator. One should consider using a self-contained or sealed ampoule BI as shown below. It can be ‘pre-placed’ in the bottom area of an empty waste bag prior to use. This would avoid at a later time, trying to place the ampoule into the bags lower portion prior to autoclaving and presenting a handling risk. A thin wire can be tied to the ampoule for easy removal after the cycle is finished. At cycle's end, pull the wire and retrieve the ampoule.

Possible waste from patients with undiagnosed or undetected HIV or hepatitis may go into the general clinical waste stream with handlers being unaware of the seriousness of the waste bag contents. A sterilization cycle failure can pose a considerable risk for handlers and the environment.

All medical/biowaste should be treated equally with a robust cycle that is easily capable of providing a minimum sterility assurance level of a log-6 spore reduction. Using a lower standard of log-4 spore reduction is just not acceptable. In order to increase our sterility confidence level from a log-4 population to a spore reduction level of log-6 may only increase our needed exposure time by a few minutes. A few additional minutes is well spent to achieve a bioburden survivor probability level of 1 in a million or a log-6 spore reduction capability. All autoclave cycles dealing with medical/lab waste should easily be capable of achieving this higher level of sterility assurance. It is this high level of performance that must be validated and uniformly applied to all medical/biowaste standards.

Russ Nyberg is director of technical support/biological indicators for Raven Labs of Omaha, Neb.


1. EPA Manual of Methods for Virology. Chapter 4, Section 6.11.

2. Born Z. Raven Labs. Waste bags and spore strips, in-house testing October 2008.

3. USP 31, SurvivalTime and Kill Time, pg. 1532.