Control Time (CT) is the incubation time used when testing samples with Delvotest® T ampoules. For each box of ampoules, CT is established using a reference sample. It is the incubation time required for the reference sample to reach the correct yellow-green endpoint colour.
A reference sample is a sample of the same matrix being tested that is expected to produce a negative result. For farm testing, reference milk should come from the complete milking of a healthy animal that has not received antibiotic treatment and is expected to produce a negative result.
Once CT has been determined, use the same incubation time when testing samples with ampoules from that box. Reading samples at the established CT helps ensure that Delvotest® T performs in line with its validated sensitivity.
Reading the test too early may cause a negative milk sample to appear positive. Reading it too late may reduce test sensitivity and increase the risk of a false-negative result.
For guidance on selecting and collecting reference milk, see ✅ Negative Reference sample for Farm Testing🔗.
Are You Using Delvotest® Go?
Delvotest® Go establishes Control Time automatically during the test procedure. You do not need to determine Control Time manually using the methods described in this article.
Recognising the Control Time Endpoint Colour
Before determining CT, it is useful to become familiar with the colour changes that occur during incubation.
The example below shows six ampoules incubated with the same reference milk. Each ampoule has been incubated for a different length of time at 5-minute intervals. Viewing several ampoules side by side makes the colour progression easier to recognise. The image below shows the colour development of Delvotest® T ampoules during incubation.
The colour typically changes from purple through the yellow-green CT endpoint to bright yellow.
The appearance of the colour may vary depending on the lighting conditions and the background against which the ampoule is viewed. This may make the colour progression and endpoint more difficult to interpret when evaluating a single ampoule, particularly when determining Control Time for the first time.
Methods for Determining Control Time
Control Time for Delvotest® T ampoules can be determined using either of the following methods:
Option 1: Using One Ampoule
Incubate a single ampoule until it reaches the yellow-green endpoint colour. This is the simplest method and is suitable for routine use.
Option 2: Using Multiple Ampoules
Incubate several ampoules at 5-minute intervals and compare them side by side. This provides a visual reference for colour development and may be helpful if you are unfamiliar with the endpoint colour.
Option 1: Determining Control Time with One Ampoule
What You Need
- 1 Delvotest® T ampoule
- Reference milk
- Disposable pipette included in the kit
- Timer
- Delvotest® T colour card
- Water bath with a lid and gentle agitation, or another suitable incubating device set and maintained at 64°C (±2°C)
Procedure
- Remove one ampoule from refrigerated storage and allow it to reach room temperature.
- Add 0.1 ml (100 µl) of well-mixed reference milk to the ampoule.
- Place the ampoule in the incubating device immediately.
- Set a timer for 2 hours and 45 minutes.
- When the timer ends, compare the lower two-thirds of the agar with the Delvotest® T colour card.
- If the agar is still purple, return the ampoule to the incubating device immediately and incubate it for another 5 minutes. Repeat this step until the yellow-green endpoint colour is reached.
- When the yellow-green endpoint is visible, record the total incubation time. This is the Control Time for that box of ampoules.
Use this CT when testing samples with ampoules from the same Delvotest® T box.
Important: Control Time may vary between ampoule batches and testing conditions. Establish the Control Time for each new box of Delvotest® T ampoules using reference milk before testing routine samples.
This method can also be performed alongside routine sample testing.
Troubleshooting
The agar is still purple
Control Time has not yet been reached. Return the ampoule to the incubating device immediately and continue incubation in 5-minute increments until the yellow-green endpoint colour is reached.
The agar is already bright yellow
The ampoule has passed the correct Control Time endpoint. Repeat the procedure using a fresh ampoule and fresh reference milk. Begin checking the colour at an earlier incubation time.
Option 2: Determining Control Time with Multiple Ampoules
What You Need
- Multiple Delvotest® T ampoules from the same box
- Reference milk
- Disposable pipettes included in the kit
- Timer
- Delvotest® T colour card
- Delvotest® MiniS, a water bath with a lid and gentle agitation, or another suitable incubating device set and maintained at 64°C (±2°C)
Procedure
- Remove only the number of ampoules needed from refrigerated storage and allow them to reach room temperature.
- Add 0.1 ml (100 µl) of well-mixed reference milk to the first ampoule.
- Place the ampoule in the incubating device immediately. Label it and record its start time.
- Repeat this process every 5 minutes for the selected number of ampoules. Prepare each ampoule immediately before incubation, label it and record its start time.
- When the most recently prepared ampoule has incubated for 2 hours and 45 minutes, compare all the incubated ampoules with the Delvotest® T colour card.
- Identify the ampoule that best matches the yellow-green endpoint colour shown in Figure 2. The incubation time of this ampoule is the Control Time for that box of ampoules.
Use this CT when testing samples with ampoules from the same Delvotest® T box.
For routine testing, including an appropriate reference sample is strongly recommended.
The example below refers to establishing Control Time with multiple ampoules. Six ampoules were incubated at 5-minute intervals. In this example, the Control Time is 3 hours and 10 minutes.
Troubleshooting
All ampoules are still purple
Control Time has not yet been reached. The batch may require a longer incubation time. Continue incubating the ampoules in 5-minute increments until the longest-incubated ampoule reaches a bright yellow colour. The preceding ampoules should then show the colour progression towards the yellow-green Control Time endpoint.
All ampoules are bright yellow
Control Time was reached before the shortest incubation time included in the series. The batch may require a shorter incubation time than expected. Repeat the procedure using fresh ampoules and fresh reference milk, and use a shorter incubation-time range.
Still Unsure About the Correct Control Time?
If you are unsure whether you identified the correct Control Time using the single-ampoule method, repeat the determination using the multiple-ampoule method. Comparing ampoules incubated at 5-minute intervals provides a clearer visual reference for identifying the yellow-green endpoint.
If you are still unsure, contact support@delvotest.com and include:
- Delvotest® product details, including the batch number
- Information about how the reference milk was collected
- The animal species and milk type
- The incubation equipment and temperature used
- Clear photographs of multiple ampoules incubated at 5-minute intervals, preferably shown alongside the Delvotest® T colour card
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