A brief history of antibiotic residue testing 

Alexander Fleming is credited with the discovery of modern antibiotics but he also made another two breakthroughs which are lesser known. These were in the area of antibiotic inhibition testing.


In 1924 he developed the ditch plate technique for evaluating the antimicrobial effects of a test solution. This involved seeding an agar with the test organism, cutting a ditch or hole within the agar, placing the test solution within the ditch and after appropriate incubation, observing for areas of inhibition.  

His second breakthrough came in 1929 when he introduced the broth dilution method which was the forerunner to the Minimum Inhibitory Concentration (MIC) method.

In 1940, it was Fleming once again who introduced yet another significant step in testing and that was the use of a pH indicator to determine changes in growth. This was the start of what we now see in all modern microbial inhibition tests, the colour changing signifying a positive or negative test result. In fact, the pH colour change is still widely used in clinical and food testing today. 

It was around this time that there was a shift from clinical testing to food and milk testing. It is hard to credit and individual for the first attempt at using a food or milk sample to see if growth was inhibited by the presence of an antibiotic. Many such developments in science go unrecorded. 

Since then further modifications were made by other scientists. They included Pope who introduced the absorbent paper in 1940, Schmith & Reyman who introduced the agar dilution (AST) and Abraham who developed the cylinder-plate method in 1941. 

In 1945 further progress was made with the impregnated disk for the susceptibility of a microorganism to antibiotic. Hoyt and Levine introduced the penicillin tablet, Moh introduced the radial streak disc method, but it was not until 1947 that a standard disk size was established. Bondi introduced the 6.5mm disk which is now the standard which we see today. 

As the usage in antibiotic became common in agriculture (since the 1950’s[1]), the FDA became concerned on its use widespread use in dairy to treat mastitis. In 1954, 1955, 1956 and 1958, the FDA conducted a survey to establish the degree of antibiotic contamination in milk. 

In one survey conducted (Pennsylvania in 1959)[8], it was found that 77% of farmers did not discard milk within the 72 hour withholding period under antibiotic treatment.

Residual antibiotics were a public health concern due to allergic reactions and toxic side effects from dietary exposure.  In addition, cultured product manufacturers would experience inhibition to their starter cultures which impacted product quality, and in some cases caused production failures. 

A nationwide survey in the USA began and revealed that penicillin was the primary antibiotic found in the central milk supply. Ten surveys covering a 9 year period (prior to 1960) in which 7,201 samples were tested, found 377 (5.2%) to be positive for the presence of antibiotics[8] 

At the same time, field testing also began and according to a New York Dairy company[8], they noticed the incidence of detection decreased 10 fold from 6.5% to about 0.5%.  This demonstrates that once people are aware that tests are happening, then common practices change for the better. 

Soon a law was introduced making it mandatory to test for antibiotics once per month. The application of testing methods by regulatory and dairy personnel during1960 resulted in a significant reduction in antibiotic-adulterated milk. Analyses ofapproximately 770,000 producer milk samples showed an incidence of 0.54% - a tenfold  reduction.  

As the amount of testing increased, it was soon noticed that the time taken for an antibiotic result was taking too long.  

In the 1960s[2], the dairy industry starting using the microbial inhibition tests that Pharmaceutical companies had developed. This reduced the test time from days to hours. 

For example, Sarcina lutea[3] Cyclinder Plate methods, and Bacillus subtilis [3][4]. Streptococcus thermophilus[5] is a starter culture microorganism that is used to this day in susceptibility studies on fermented products.

In the late 1970s, microbial inhibitions tests employed by the dairy industry utilized a more sensitive microorganism, Bacillus stearothermophilus [6][7].  It was not until the late 70’s when the breakthrough came. That was the receptor based assay.   


Receptor based technology (Results in minutes, not hours) 

It was 1978 when Dr Stanley Charm, a professor at the Massachusetts Institute of Technology and President of Charm Sciences, launched the world’s first receptor assay method for antibiotic testing.  

The outcome was the Charm I, which was at the time, the world’s first test to provide a numerical result for beta-lactam in less than 15 minutes. Unlike the broad spectrum microbial inhibition test, the Charm I offered greater speed, sensitivity and specificity to beta-lactam antibiotics. 

In the dairy industry, this allowed raw milk tankers which would normally queue up at tanker bays for hours waiting for a clearance a quicker test result. The Charm I started a revolution and gave the dairy industry the ability to control their raw milk supply by providing a result in minutes than hours allowing milk to be unloaded quickly. 

In 1986, the Charm II system was introduced and the testing time was reduced from 15 to 10 minutes.

The real capability came about when multiple antibiotics families were able to be detected which quickly achieved AOAC approval for 7 families of antibiotics. It became the choice instrument for regulatory testing around the world. 

As speed became the norm within the industry, Charm sparked another new revolution in dairy testing with its ROSA (Rapid One Step Assay) platform. The patents can be found here. 

Unlike most rapid systems available which involve at least two to three steps - the ROSA system simplified it to just one,  making it simple and easy to use. With a reduction in the number of steps, fewer mistakes are made leading to a reduced risk of rejecting ‘good’ milk through poor operator error.  

Test time is 8 minutes and there is now even a 3 minutes test making it the quickest test available. 

But what sets Charm apart from other kit suppliers is that they continue to add value. The system includes data acquisition software with the ability to link it to LIMS system such as SAP EEC6.  

Charm tests also have the ability to incorporate RFID / Key FOB integration to strengthen data security. And with bar-coded test strips there is less chance of a rogue operator falsifying a test result just to load in milk. 

And it doesn’t stop there – whilst most test strips on the market only test for beta-lactam and maybe even tetracyclines. The Charm ROSA system has expanded its range to include beta-lactams, tetracyclines, sulfonamides, chlorophenicol chloramphenicol, enrofloxacin, streptomycin and even aflatoxin M1 making it the preferred choice for the top global dairy companies. 

Stay tuned for my next issue on what dairy farmers are using on the farm level. This will be sent mid-January so you can have a break for now!  


Best wishes for Christmas and hope you have a prosperous New Year! 


Arrow Scientific Pty Ltd

Unit 1, 332 Burns Bay Road, Lane Cove NSW 2066

Ph: 02 9427 7455  -  Fax: 02 9427 7456  -   www.arrowscientific.com.au 


1 Quantitative Assay for Antibiotics Used Commonly in Treatment if Bovine Infections. 1985 Journal of Dairy Science 68: 3031-3036

2 http://jds.fass.org/cgi/reprint/46/2/95.pdf

3 Evaluation of two microbiological methods for detecting residual antibiotics in milk. Journal of the Association of Analytical Chemists. 1976 Sep;59(5):1122-24.

4 Detection of Sulfa Drugs and Antibiotics in Milk. APPLIED MICROBIOLOGY, May 1971, p. 806-808, Vol. 21, No. 5

5 A simple test for the detection of antibiotics and other chemical residues in ex-farm milk.  Food Control, Volume 10, Issue 1, February 1999, Pages 35-39

6 IDF 1970. Detection of Penicillin in Milk by a Disk Assay Technique. International Standard FIL-IDF 57. Brussels).

7 Diffusion Test for the Determination of Antibiotic Residues in Milk. Neth. Milk and Dairy J. 29 (1975)

8 Albright, J. L., Tuckey, S. L., Woods, G. T. “Antibiotics in Milk A Review” Journal of Dairy Science. (1961) 44: 779-807


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