Acidification test for phage detection (Danisco)
TM 2066-2e
EFFECT
The acidification test is used for the indirect detection of phages in a sample. A bacterial culture, both with and without the sample believed to carry phages, is incubated in milk. After incubation, the pH is determined. A delay in the acidification of the test sample compared to the control sample indicates a bacterio-phage infection.

MEDIA, REAGENTS, EQUIPMENT
Sterile milk medium pH meter
Glass funnel
Folded paper filter
Disposable syringe with disposable sterile filter, pore size <0.45μm
Sterile glass tubes (for acidification test and sample storage)
Sterile lactic acid (~20%)
Sterile 1.8ml cryo tubes

SAMPLE PREPARATION
• Place a folded paper filter in the glass funnel
• Mix the sample well
• Acidify (pH 4.5-4.8) using sterile lactic acid if necessary
• Separate the solid components in the sample from whey using the folded filter
• Hold the filtrate with a disposable syringe
and press through the disposable sterile filter at a constant pressure
• Use the sterile filtrate for the phage test

PROCEDURE
Setting up the culture pool for the test
• Choose the cultures to be tested
• For bulk starter cultures
- Prepare a bulk starter in milk and incubate at 24°C (mesophilic cultures) or 37°C (thermophilic cultures) overnight until set
- Dilute the prepared bulk starter with sterile milk at a ratio of 1:10 and freeze in sterile 1.8ml cryo tubes at -25°C to -45°C (maximum storage 6 months)
• For direct starter cultures
- Dilute the culture in sterile milk (dilution 50u Direct FP/2L; 10u Direct FD/1L); and ensure the culture is fully dissolved and well mixed
- Freeze the diluted culture in sterile 1.8ml cryo tubes at -25°C to -45°C (maximum storage 6 months)
• Deposit the number of cryo tubes that are expected to be required over a given period
• Always use a new culture batch when renewing the pool

Preparation of cultures for the test
• Use a freshly prepared culture from the pool for every new acidification test. The sub-cultivation of cultures can cause modification of the strains and, thus, produce false results
The acidification test
• For bulk starter cultures
- Add approx. 1ml of the culture from the cryo tube to 9ml of sterile milk and incubate overnight at 24°C (mesophilic cultures) or 37°C (thermophilic cultures)
- Dilute the fermented culture with sterile milk at a ratio of 1:10 and use 1ml of the dilution to inoculate the 9ml sterile milk tubes for the acidification test (1% inoculum; milk temperature at time of inoculation 4-10°C)

• For direct starter cultures
- Dilute the culture from the cryo tube in sterile milk at a ratio of 1:10 and use 1ml of the dilution to inoculate the 9ml sterile milk tubes for the acidification test (1% inoculum; milk temperature at time of inoculation 4-10°C)
• Use one batch of milk for each trial series
• Heat some of the sterile filtered sample that is to be phage tested for 10min at 90°C
• Add 1% of the unheated sterile filtrated sample to a 9ml milk tube, 1% of the heated sterile filtered sample to another
9ml milk tube and mix thoroughly
• Prepare the reference without adding the sterile filtrated sample (just the culture)
Culture Sterile filtrated sample
reference unheated sterile filtrate heated sterile filtrate
milk tube sample milk tube sample milk tube
Danisco A/S
Edwin Rahrs Vej 38
DK-8220 Brabrand, Denmark
Telephone: +45 89 43 50 00
Telefax: +45 86 25 10 77
info.ingredients@danisco.com
www.danisco.com
The information contained in this publication is based on our own research and development work and is to the best of our knowledge reliable. Users should, however, conduct their own test to determine the suitability of our products for their own specific purposes. Statements contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for the infringement of any patents.

02.05
• Incubate the inoculated milk tubes as follows:
- Mesophilic cultures : ~6h; 30°C; final pH (reference) <5.6
- Thermophilic cultures : ~2-4h; 42°C; final pH (reference) <5.6
• After incubation, check if the final pH corresponds to the reference
• If necessary, extend the incubation time
• Note final pH of the reference and the sample

ENRICHMENT
In the case of very low phage levels and/or to ensure the reliability of the result, an enrichment step is helpful. During the second passage low amounts of phages will be enriched and may lead to a more significant acidification failure. Inhibiting substances will be diluted further, reducing
the probability of false positive phage results. In this way, low titers of phage may also be detected.
Procedure: For this, the first acidification test with the unheated sample is incubated further after measuring the pH (in order to avoid contaminating the sample with the pH probe, measure pH in the tube cap and discard – see waste disposal) until whey is formed. This whey is then filtered through the paper and sterile filters, and a second acidification
test is conducted with the new sterile sample.

RESULTS AND EVALUATION
If the acidification is delayed in the sample by more than 0.05 pH units compared to the reference, the presence of phage is assumed. Phage should, in general, be dead in the heated sample. If the acidification of both samples is delayed, it is assumed that there are other inhibitors than phage in the samples. But it should be remembered that there are also heat-sensitive inhibitors. So there is a possibility of other inhibitors if the heated samples show no delay.

SAFETY NOTES/WASTE DISPOSAL
After the test, the tubes and all other material that has been in contact with the samples should be cleaned/soaked using a chlorine solution (200ppm) and autoclaved in the phage laboratory to avoid cross-contamination. As the pH probe cannot be cleaned with chlorine or autoclaved, it should not be used for tests other than the acidification test in order to avoid spreading phage in the lab