🚧 Establishing Control Time

Delvotest® is a microbial inhibition test that can detect antibiotics at certain detection limits as described in the sensitivity lists. The detection described on these lists has been established by reading the test at the correct color changing moment, the so called "Control time". 

Under the correct incubation conditions, Delvotest® will start changing color from purple to yellow within a few hours. (Read more on the working principle of Delvotest in this knowledge article). The control time (CT) is the moment during incubation when the test has just turned yellow when testing with negative control milk, without antibiotics. But the CT is not always the same. It is changing periodically and for this reason it is important to understand how to establish it and when to read at control time. 

This section below explains;

• The importance of testing at Control time 
• Expected control time regions per test type and animal species 
• Procedure to find the control time with ampoules as an example

Why ‘Control Time’

At control time the sensitivity of the test is optimal. When incubated longer than the control time the sensitivity of the test is decreased. Reading at fixed time of 3 hours 15 minutes for Delvotest® T or 3 hours for Delvotest® SPNT is an example where the sensitivity can be reduced. 

Let's take a look on the example below: 

Two ampoules were prepared according to the Delvotest® instructions, one ampoule contains known negative milk control and the other contains a milk sample with the antibiotic tetracycline above the detection limit. After incubation, we read the results. 

• Reading at control time gives us the expected results. When the milk without antibiotics is turning yellow, the sample with tetracycline has a slightly purple color and is positive. 
• If we incubate the ampoules longer than the control time, we notice that the negative milk sample has now a very bright yellow color and the sample with antibiotics gives a negative result. The sensitivity was reduced because the ampoules were over incubated. 

When to to use 'Control time'

Due to the nature of Delvotest®, the exact moment of color change varies slightly from batch to batch and throughout the shelf life. To make sure you test with the best sensitivity, it is necessary to verify the CT of your product periodically. 

The control time needs to be established separately also for different animal species, for example goat and sheep because they have milk with different composition than cow. 

Here are some cases when to test with negative control milk:

• When you want to test with the best sensitivity 
• When you first receive your Delvotest® box or
• When you test with a new batch of Delvotest®
• When you use your Delvotest® more than 30 days 
• When you use milk from different animal or different dairy matrix

To optimize reliability, dsm-firmenich advises to verify the correct functionality of the test, operations and incubation with negative control and positive control milk samples available at dsm-firmenich. 

Expected Control time per test and species

Before testing for control time

Delvotest® microbial inhibition tests, work with the presence of a milk sample. Use this guide here for more details on the correct Delvotest® sample preparation.

Finding Control time-using one ampoule with negative milk 

• After opening the aluminum foil, pipet in one ampoule a negative control 0.1 ml (100 μl ± 10 μl). Incubate immediately at 64 ± 2°C.
• To verify the correct control time (CT) set the timer on the following incubation time: 
  • 3h:00m for Delvotest® T.
  • 2h:30m for Delvotest® SP NT
• After the incubation time is over, remove immediately the ampoule from the incubator and immediately check the test colors using the color card. 

The correct control time (CT) is when the color of the antibiotic free milk sample is between the colors --- and – as indicated on the color card. 

Delvotest® T ampoules at control time- Digital reference

Delvotest® SPNT ampoules at control time - Digital reference

• When the color is too yellow (--- on the color card): repeat the test shortening the incubation time by 5 minutes. Repeat the exercise, shortening every time the incubation by 5 minutes, until the correct color is achieved.
• When the color is still too purple (+ or +++ on the color card): IMMEDIATELY return the ampoule into the incubation unit and incubate for 5 minutes more. Repeat the color check at intervals of 5 minutes, until the correct color is achieved.
• Register the control time (CT) found and use this time for the incubation of the ampoules of the same batch. Incubation conditions must be the same in a dry incubator or water bath.

Procedure for Control time estimation using five ampoules

1. Select 5 ampoules. 
2. Mark the time on each ampoule in 5 minutes incubation time. If you determine an approximate Control Time at around; 03h:00m for Delvotest® T, and 02h:30m for Delvotest® SP NT, using 5 ampoules, mark the ampoules as:
   • For Delvotest® T at around 03h:00m, select 2h:50, 2h:55, 3h:00, 3h:05, 3h:10.
   • For Delvotest® SP NT at around 02h:30m, select 2h:25, 2h:30, 2h:35, 2h:40, and 2h:45.
3. After opening the aluminum foil, pipet in one ampoule a negative control (100 μl ± 10 μl) and incubate immediately at 64 ± 2°C.
4. Set the timer on the incubation time of the first ampoule (the one incubated the longest, for Delvotest® T 3h:10 or for Delvotest® SPNT 2h:45 in the example at point 2a and 2b).
5. Pipet and incubate the remaining selected ampoules, one every 5 minutes, taking into consideration the time that takes between preparing one ampoule and the start of the incubation. It’s crucial to respect the times. Meaning next ampoule must be pipetted shortly before to insert it into the heating device
6.  After the longest incubation time (Delvotest® T 3h:10 or for Delvotest® SPNT 2h:45) is over, proceed immediately removing all the ampoules from the incubator. If the ampoules cannot be read immediately, cool them in cold/iced water for 5-10 minutes, to stop the incubation procedure. Below an example (Delvotest® T ---, -, +, +++) of results when using 4 ampoules. 
7. Evaluation using the color card as explained in the next section.