| Department | Micro Laboratory | Document no | MICLAB 065 | ||
| Title | Determination of Heat Resistance of Spore Forming Organisms | ||||
| Prepared by: | Date: | Supersedes: | |||
| Checked by: | Date: | Date Issued: | |||
| Approved by: | Date: | Review Date: | |||
Document Owner
Micro Laboratory Manager
Affected Parties
All Microbiology Laboratory colleagues
Purpose
To describe the method for calculating the Heat Resistance Factor, (D-value),of spore-forming organisms.
Scope
This SOP is to be followed by all Microbiology Laboratory staff for determining the D-value of a spore-forming organism.
Definition
| D-value | Is defined as the time required for a population of a pure culture of microorganisms to be decreased by 90% when exposed to a fixed temperature, e.g. 121°C (±1°C). |
Related Documents
| MICLAB 055 | Microbiological Monitoring of Plant Water Systems |
| MICLAB 075 | Micro Evaluation on Bioburden, Non sterile and Raw Materials |
EHS Statement
Safety Glasses must be worn when heat sealing an ampoule and when using the Constant Temperature Bath set at 121°C (±1°C).
Procedure
1. If the spore‑forming organism is an aerobe subculture it onto a Trypticase Soy Agar (TSA) and Nutrient Agar (NA) or Sporulation Agar (SA) slope at 32°C (±1.5°C) for 48 hours. If the spore-forming organism is an anaerobe grow it on a Reinforced Clostridium Agar slope in an anaerobic jar at 32°C (±1.5°C) for 3 days.
2. Using the spore stain described in Step 3, ensure that sporulation has taken place in 50% or more of the culture population.
3. Spore Stain ‑ Schaeffer & Fulton
3.1. Flood the slide with 2% aq. Malachite Green.
3.2. Heat the slide to steaming for 5 minutes over a small beaker of water.
3.3. Remove from heat and leave the Malachite Green solution to act for 10-15 minutes.
3.4. Rinse under running water.
3.5. Counterstain with 0.5% aq. Safranin for 30 seconds or Carbol Fuschin for 15-20 seconds.
3.6. Rinse with water and drain or blot to dry.
Bacterial bodies stain Red; Spores stain Green.
4. If sporulation has taken place in 50% or more of the population proceed to step 5. If sporulation is <50%, re-incubate the culture at 37°C (-0.5 to +1.5°C) and examine daily for sporulation. The culture can be incubated for up to 1 week at 37°C (-0.5 to +1.5°C).
If sufficient sporulation is not achieved after this time, return to Step 1.
5. Wash the growth from the slope with 5-10mL sterile Distilled Water into a sterile McCartney Bottle. Spin down the culture at 4000rpm for 10 minutes. Remove the supernatant and re-suspend the pellet in 10mL sterile Distilled Water. Repeat this step twice.
6. Heat the suspension using an Attest Biological Incubator at 56°C (±1°C) for 30 minutes to kill any vegetative organisms that may be present.
7. Transfer 3mL of the spore suspension to a 5mL glass ampoule. Heat‑seal the opening without heating the suspension by using tongs and a Bunsen burner. This ampoule will be used in Step 8 and the remaining spore suspension can be left in the McCartney bottle for use in Step 9.
8. Calculation of Final Number of Spores Present per mL
8.1. Using a pair of tongs, submerge the 5mL ampoule for 2 to 5 minutes in the Constant Temperature Bath set at 121°C (±1°C).
8.2. Remove the ampoule and plunge it into a beaker of iced water.
8.3. Perform serial dilutions to 10-2 using 9mL volumes of peptone water in autoclaved McCartney bottles.
8.4. Using the Plate Count Method, obtain a final spore count on the cooled suspension. Perform spread plates on Nutrient Agar using 0.1mL of the 100, 10-1 and 10-2 dilutions and incubate at 32°C (±1.5°C) for 24-48 hours.
8.5. Select the plate that yields between 20-200 colonies and calculate the number of spores remaining in cfu/mL. This value is used as part b of the D-value calculation in Step 10.
9. Calculation of Initial Number of Spores Present per mL
9.1. Using the Plate Count Method, obtain an initial spore count on the remaining spore suspension from Step 7. Perform serial dilutions to 10-6 using 9mL volumes of peptone water in autoclaved McCartney bottles. Store the remaining spore suspension at 4°C (±1°C) until the D-value determination of the organism is complete.
9.2. Perform spread plates on Nutrient Agar using 0.1mL of the 10-3 to 10-6 dilutions and incubate at 32°C (±1.5°C) for 24-48 hours.
9.3. Select the plate that yields between 20-200 colonies and calculate the number of spores present in cfu/mL. This value is used as part a of the D-value calculation in Step 10.
10. Calculation of Heat Resistance Factor (D-value)
10.1. Substitute the values calculated for a and b into the following equation to calculate the
D-value of the organism.
10.2.
u = D (log10 a-log10 b)
Therefore:
D =
u
—————–
log10 a-log10 b
Where,
u = heating time (minutes).
a = initial number of spores present per mL.
b = final number of spores present per mL.
11. Check the D-value limit requirements for the sample in which the spore-forming organism was isolated. For Distilled Water samples, see MICLAB 055. and for Filled Container Bioburden samples, see MICLAB 075. Record the calculated D-value into work book or log book. If the D-value determined exceeds the stated limit, immediately inform the Microbiology Manager.
Summary of Changes
Version # | Revision History |
MICLAB 065 |