Horizontal method for the enumeration of microorganisms - Part 1: Colony count at 30°C by the pour plate technique
ISO 4833-1 (2013) method: Colony Count
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1 Quantitative process control with CRM-SA
- Use a quantitative reference material (CRM-SA) with a known number of Staphylococcus aureus.
- Defrost the material at room temperature for 30 minutes.
- Mix well before use and dilute the material so countable numbers are inoculated on the media (MPCA) by pour plate method.
- Best results are obtained when a practicable range of 80 cfu to 120 cfu per plate with a minimum number of 50 cfu per plate is used.
- Incubate at (30 ± 1) °C for (72 ± 3) h.
- Count the colony forming units and register in a control chart.
- The characteristic reaction is: Growth.
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2 Quantitative process control with RM-BLES
- Use a quantitative reference material (RM-BLES) with a known number of Microorganisms.
- Defrost the material at room temperature for 30 minutes.
- Mix well before use and dilute the material so countable numbers are inoculated on the media (MPCA) by pour plate method.
- Best results are obtained when a practicable range of 80 cfu to 120 cfu per plate with a minimum number of 50 cfu per plate is used.
- Incubate at (30 ± 1) °C for (72 ± 3) h.
- Count the colony forming units and register in a control chart.
- The characteristic reaction is: Growth.
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3 Quantitative process control with RM-BSUBT
- Use a quantitative reference material (RM-BSUBT) with a known number of Bacillus subtilis subsp. spizizenii.
- Defrost the material at room temperature for 30 minutes.
- Mix well before use and dilute the material so countable numbers are inoculated on the media (MPCA) by pour plate method.
- Best results are obtained when a practicable range of 80 cfu to 120 cfu per plate with a minimum number of 50 cfu per plate is used.
- Incubate at (30 ± 1) °C for (72 ± 3) h.
- Count the colony forming units and register in a control chart.
- The characteristic reaction is: Growth.
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4 Quantitative process control with RM-CC
- Use a quantitative reference material (RM-CC) with a known number of Microorganisms.
- Defrost the material at room temperature for 30 minutes.
- Mix well before use and dilute the material so countable numbers are inoculated on the media (MPCA) by pour plate method.
- Best results are obtained when a practicable range of 80 cfu to 120 cfu per plate with a minimum number of 50 cfu per plate is used.
- Incubate at (30 ± 1) °C for (30 ± 1) °C.
- Count the colony forming units and register in a control chart.
- The characteristic reaction is: Growth.

Best Practice
The single strain CRM-SA is used for the enumeration of Coagulasepositive staphylococci. In accordance with ISO 4833-1, PCA can also be used instead of MPCA.

Best Practice
The multi-strain RM-BLES contains, Bacillus cereus, Lactobacillus plantarum, Escherichia coli WDCM 00013 and Staphylococcus aureus WDCM 00034 in milk. This material can be used for several specific standards. The influence of interfering bacteria is also performed. It is easy to use by making one serie of dilution.
Kind of reference material | Biosisto product for this control method | Control Strain | WDCM number | NCCB number | |
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Multi-strain reference material | Bacillus cereus | 100292 | |||
Lactobacillus plantarum | 100293 | ||||
Escherichia coli serotype O6:H1 | 00013 | 100297 | |||
Staphylococcus aureus | 00034 | 100294 | |||
The Biosisto reference material in this table is used in the best practice for Quantitative process control according ISO 17025. |

Best Practice
The single strain RM-BSUBT containing vegetative cells and heat-resistant spores in milk. This material can be used for analyses cell and spore of Bacillus subtilis as well for Colony count analyses.
Kind of reference material | Biosisto product for this control method | Control Strain | WDCM number | ||
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Single-strain reference material | Bacillus subtilis subsp. spizizenii | 00003 | |||
The Biosisto reference material in this table is used in the best practice for Quantitative process control according ISO 17025. |

Best Practice
The multi strain RM-CC that contains, Staphylococcus aureus WDCM 00034, Bacillus subtilis WDCM 00003 and Escherichia coli WDCM 00012 in broth. This material can be used when the proces must be controlled with interfering bacteria or if you want to use this material for multi purposes.
Kind of reference material | Biosisto product for this control method | Control Strain | WDCM number | NCCB number | |
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Multi-strain reference material | Echerichia coli | 00012 | 72002 | ||
Staphylococcus aureus | 00034 | 100294 | |||
Bacillus subtilis | 00003 | 89157 | |||
The Biosisto reference material in this table is used in the best practice for Quantitative process control according ISO 17025. |