Growth Control, Positive Control and Incubation Temperature in Spore Testing

CliniEco 24-hour biological indicator incubator, 15-well precision dry block for spore testing

A growth control - usually written as positive control - is a biological indicator that has not been sterilized, incubated alongside the processed indicator to prove the test itself worked. It is the single most misunderstood step in clinic spore testing, because a clinic that reads only the processed indicator can record a negative result that means nothing. This article sets out what the control is, what it proves, why it must come from the same lot code, what incubation temperature each process requires, and what to do when the control and the test disagree.

CliniEco 24-hour biological indicator incubator, 24-well precision dry block for spore testing

Quick facts

  • The control is a non-processed indicator that must grow. If it does not show growth, the run is invalid - a negative test result alongside a negative control is not a pass, it is an unreadable test.
  • One control is incubated each day a processed indicator is incubated, from the same lot code as the processed indicator, labelled with a "C" and the date, according to manufacturer instructions for use.
  • Saskatchewan makes the control a written requirement in its own right. The CDSS infection prevention and control standards state that one control biological indicator must be incubated each day to confirm that the incubator is functioning, and that the control's date and time must be recorded and signed.
  • Incubation temperature is set by the process, not by convenience. Steam indicators are incubated in the 55 to 60 C range, with 57 C and 60 C both published on commercial dry-block incubators; ethylene oxide indicators are incubated at 37 C.
  • ISO 11138-1:2017 section 7 covers culture conditions for biological indicators, so incubation temperature and medium requirements have a standard basis rather than a vendor convention.

What is a growth control in spore testing

A growth control is an unprocessed biological indicator from the same production lot as the test indicator, activated and incubated at the same time and in the same incubator. Its expected result is the opposite of the test indicator's: it must show growth - a positive result - because nothing killed the spores it contains.

That inversion is the whole point. The processed indicator's negative result is only meaningful if the system that produced it can still detect a live spore. The control demonstrates four things at once, as set out in the instructions for use for a widely used super rapid steam indicator:

  1. that incubation temperatures were actually met;
  2. that the spores' viability was not altered by improper storage temperature, humidity or proximity to chemicals;
  3. that the culture medium is capable of promoting growth;
  4. that the incubation process itself is functioning.

Ontario guidance reaches the same requirement from the regulatory side - a control test must also be used when spore testing is performed, and those results also recorded. Saskatchewan's CDSS standards state it directly: one control biological indicator must be incubated each day to confirm that the incubator is functioning, and the control should yield positive results for bacterial growth, with its date and time recorded and signed.

Item Processed indicator Growth control (positive control)
What it is A biological indicator that went through the sterilization cycle A non-processed indicator from the same lot code
Purpose Test whether the cycle killed the spores Prove the test system can detect a live spore
Expected result Negative (no growth, no fluorescence) Positive (growth, fluorescence)
Placement Inside the load, in the process challenge device or area least accessible to steam Not in the sterilizer at all
Labelling Cycle or load identifier "C" plus the date
If it deviates Investigate the cycle as a failed process Treat the run as invalid and repeat it
Record kept Date, sterilizer, cycle, result Date, time, result, signature of the responsible practitioner

Why must the control come from the same lot

Because the control is testing the indicator, not the sterilizer. Lot-to-lot variation in spore population, carrier material and medium is exactly what the control is meant to catch, so a control from a different lot proves nothing about the indicators in use. The instructions for use for a super rapid steam indicator specify that the positive control should be from the same lot code as the processed biological indicator; where a clinic holds one box at a time this is automatic, and where two boxes are open it is not.

The practical rule is to keep one box open. When a new lot is put into service, that lot's first control run is the moment to confirm the lot behaves as expected, and the lot code belongs in the record so the pairing can be reconstructed later.

What incubation temperature does my spore test need

Incubation temperature follows the test organism, and the test organism follows the sterilization process. Interchanging incubators between processes is the error to avoid, because a steam indicator in an ethylene oxide incubator is not a slow test - it is a meaningless one.

Sterilization process Test organism in common supply Incubation temperature Applicable indicator standard
Moist heat (steam) Geobacillus stearothermophilus 55 to 60 C, with 57 C and 60 C both published by incubator manufacturers ISO 11138-3:2017
Ethylene oxide Bacillus atrophaeus 37 C ISO 11138-2:2017
Dry heat Bacillus atrophaeus Per indicator instructions ISO 11138-4:2017
Low-temperature steam and formaldehyde Per indicator instructions Per indicator instructions ISO 11138-5:2017
Vaporized hydrogen peroxide Per indicator instructions Per indicator instructions Part 6 is in draft; ISO 22441 and ISO 14937 address the process
General requirements across all parts Set by the indicator manufacturer Covered by culture conditions in section 7 ISO 11138-1:2017

Dry-block incubators in clinic use publish their own temperature set points: a 24-well unit is sold as a precision dry block for 24-hour indicators, an 11-well aluminium block is published as operating at 55 to 60 C, a 10-well unit at 57 C, and single-process units at 60 C for steam and 37 C for ethylene oxide. Two specifications decide whether a block suits a clinic's indicators, and neither is the well count: well diameter must match the indicator tube - 11 mm and 13 mm blocks take different bottle families, and a 13 mm bottle does not fit an 11 mm well - and the block's temperature must match the process. A block running at 37 C cannot incubate a steam indicator, whatever the well count.

CliniEco Medical 24-hour self-contained steam biological indicators, 25-pack

What do I do when the control and the test disagree

There are four combinations, and only one of them is a pass.

Processed indicator Control result Interpretation Action
No growth Positive (growth visible) Valid test; cycle passed Release the load per policy
Growth Positive (growth visible) Valid test; cycle failed Treat as a process failure: quarantine the load, investigate, re-test before returning the sterilizer to use
No growth No growth Invalid test The result cannot be read; repeat the control and the test. Check incubator temperature, indicator storage and expiry first
Growth No growth Invalid or anomalous Investigate the control conditions and the lot; do not release the load on either result

The third row is the one clinics get wrong. A negative test with a negative control looks like two good results on a monitoring sheet, and it is actually a failed test with no information about the cycle. Recording it as a pass is the single most common way a monitoring record loses its evidential value.

Manufacturer instructions add a further instruction on the positive side: if a processed indicator is positive, identify the cause following facility policies and procedures, re-test the sterilizer, and do not use it for processing loads until consecutive negative results are obtained.

What belongs in the record

The control requirement is a documentation requirement as much as a laboratory one, because an inspector sees the record and not the bench. What the source documents ask for:

  • The control's date and time, with a signature. The CDSS standards require the control indicator's date and time to be recorded and signed by the responsible oral health care professional.
  • The result of the control, not only of the test. RCDSO guidance states that a control test must also be used when spore testing is performed and those results also recorded.
  • The identifying detail that makes the record auditable - sterilizer, date, cycle and the person completing the process, which the same provincial standards list as the minimum content of in-house biological monitoring records.
  • Lot codes and expiry dates for both the processed indicator and the control, so that a later recall or investigation can be traced back.

For a clinic setting the routine up, a biological indicator 5-pack trial covers the first week with controls, a 24-hour self-contained 25-pack is roughly a month of daily monitoring for one sterilizer once the control is counted in, and a 24-well dry-block incubator sized as "daily tests plus one control well" avoids the common shortfall where the block has exactly as many wells as the clinic runs tests. Clinics reading fluorescence results in-session use the 3-hour fluorescence indicator with a matching reader.

Does a control replace anything else

No. The control sits inside the spore testing workflow; it does not replace the daily biological indicator itself, the external and internal chemical indicators on each package, the air removal test at the start of the day on a dynamic air removal sterilizer, or periodic sterilizer maintenance and calibration. It also does not validate a new lot of indicators beyond the run it accompanies - it is a same-day check, repeated every time a processed indicator is incubated.

Sources

Ordering for a clinic, lab or care home? Wholesale and multi-site ordering covers case pricing and account setup, and the B2B wholesale collection lists the lines stocked for institutional buyers.

Related reading

The failure side of the same workflow is worked through in our spore test FAQ on incubation and failed results, and the bench-level routine is set out in spore test incubator instructions: 57 to 60 C, 24 hours, read and record. Temperature and capacity choices across the market are compared in our biological indicator incubator guide, and the full monitoring chain - physical, chemical and biological - is mapped in the sterilization compliance hub.

Related Reading

Frequently Asked Questions

What is a positive control in spore testing

It is a biological indicator that has not been sterilized, from the same lot code as the processed indicator, activated and incubated alongside it. Its expected result is growth, because nothing killed its spores. It confirms that incubation temperature was met, that spore viability was not damaged by storage conditions, and that the medium promotes growth - which is what makes the processed indicator's negative result readable.

What happens if the control does not grow

The test is invalid and cannot be read as a pass. A negative processed indicator alongside a negative control means the system failed to demonstrate that a live spore would have been detected. Check the incubator temperature, the indicator storage conditions and the expiry date, then repeat the control and the test. Do not release the load on either result.

Why must the control come from the same lot as the test indicator

Because the control tests the indicator as well as the incubator. Spore population, carrier material and medium can vary between lots, so a control from a different lot does not demonstrate that the lot in use would respond. Manufacturer instructions for a widely used super rapid steam indicator specify that the positive control should be from the same lot code as the processed biological indicator.

What temperature should a steam spore test be incubated at

Steam indicators based on Geobacillus stearothermophilus are incubated in the 55 to 60 C range, and commercial dry-block incubators publish set points including 57 C and 60 C. Ethylene oxide indicators use a different organism and are incubated at 37 C. Never incubate a steam indicator at 37 C or an ethylene oxide indicator at 60 C; the temperature matches the organism, and the organism matches the process.

Do I need a control every day or only when I test

A control is incubated each day that a processed indicator is incubated. Saskatchewan's CDSS standards go further and require one control biological indicator to be incubated each day to confirm the incubator is functioning, with the date and time recorded and signed. In routine daily monitoring the two coincide, so the practical answer is one control per day of testing.

How many incubator wells do I need for controls

Plan the block as daily tests plus at least one control well, and check the well diameter against the indicator tube before ordering. An 11 mm block and a 13 mm block accept different bottle families. A clinic running three daily tests with one control needs four positions in use, which is why the control is counted in the capacity calculation rather than treated as spare capacity.

Is a chemical indicator a form of control

No. A chemical indicator responds to cycle variables such as time, temperature and steam, and cannot show spore survival. The positive control is a biological indicator containing live spores. Both belong in the monitoring record, and neither substitutes for the other.

CliniEco Medical distributes in Canada under MDEL #35334.

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