Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
Volume 23
Issue 2
Food Science and Technology
DOI:10.30825/5.ejpau.188.2020.23.2, EJPAU 23(2), #04.
Available Online: http://www.ejpau.media.pl/volume23/issue2/art-04.html


Jakub Biegalski
Department of Dairy Products Quality, Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Poland



Nowadays, cup with a drink is an attribute of a modern consumer. It is used for hot drinks (e.g. coffee, tea, hot chocolate) and cold drinks (e.g. fruit juices). Currently, there is a noticeable tendency to replace disposable cups with one reusable cup. The barrier to their reuse (especially the mouthpiece on the lid), is the lack of knowledge about its microbiological state. The aim of the presented research was to estimate the risk of bacterial etiology occurrence on disposable and reused lids. The overall metabolic activity of microorganisms was assessed by measuring ATP using the bioluminescence method. Detection of microorganisms was carried out on the abiotic surface (PP / PS), both on the lids of disposable cups used once and repeatedly. Contact with the personnel's hands was also considered. The results of the experiment indicate that the contact of the lids with the personnel's hands is not a reason for the increasing amount of ATP. The highest amounts of ATP were observed on lids of cups used repeatedly.

Key words: Bioluminescence, Abiogenic surface, Takeaway cups, Consumer health.


Cup with a drink is an attribute of a modern, active and mobile consumer. Cups are usually used for hot drinks, such as coffee, tea, hot chocolate or cocoa. They are also used for cold and iced beverages / drinks, for example shakes, lemonades, fruit juices and crushed ice drinks. Consumers are usually using disposable, “take away” cups. Beverages dispensed in this kind of cups are offered primarily at fast-food restaurants, bars, shopping center cafes, canteens, gas stations and self-service vending machines. The material from which the cup is made, depends on the temperature of the drink. Nowadays, polypropylene (PP), polyethylene (PE), polystyrene (PS) and polyvinyl chloride are used. Disposable cups are characterized by various shapes and thicknesses.

Consumer are increasingly aware of sustainable development in environment, human sustainable behavior, pro-ecological behavior, huge amounts of unrecyclable waste after just minutes of utility and the fashion for a lifestyle known as “Zero Waste”. Currently, global drive is to reduce the burden of plastic waste in the environment.

There is already a tendency to replace several disposable cups with one reusable cup. Consumers are often encouraged to get a coffee discount if they bring their own reusable cup.

There are more and more actions and slogans in public encouraging to switch from “to-go” cups to a sustainable alternative. David et al. [2] showed, that in just a couple of weeks, with dynamic-norm intervention for sustainable consumption, helps customers avoid disposable cups, and increases their use of reusable alternatives by 17.3%. Reusable cups with a possibility of closing or with protective lid with a mouthpiece to facilitate drinking and to prevent spilling drink, e.g. in a bag or backpack, are used.

In the case of disposable cups, many of them also have lids. The construction of the lid enables putting/pressing it on the cup. It is used for mechanical and microbiological protection of drink inside. It is also a preventive barrier that maintains the temperature of the drink. Research on time of cooling the coffee in reusable thermoplastic cups with and without lid was conducted by Naik et al. [3].

The lids that are most common when buying a take-out hot drink are equipped with a profiled mouthpiece and vent hole. They also have various types of graphics, patterns, buttons and holes. The most popular are cup lids for a cups with a nominal capacity from 200 to 450 mL and a ratio of nominal capacity larger than 80%. Their diameters ranges between 80 and 90 mm, and thickness between 1.0 and 2.5 mm. Both cups and lids  are usually packed in polyethylene sleeves, and then closed by welding or putting on a wire clip. In this way, they reach gastronomic points or vending machines. They are not subjected to additional disinfection procedures. After pouring the drink into the cup, the lid is put on. This procedure is done manually by the consumer or catering establishment staff. Hands can be a source of bacterial infection/reinfection.

Observations of this activity prompted to undertake a research on the actual cleanliness of the lids/mouthpieces before connecting with mouth and drinking a drink from them. A much more serious bacteriological problem is the cleanliness of the lid/mouthpiece of reusable cups. They can be a reservoir of microflora, diverse in species and quantity, and thus they can be a reason of infection. This will be clearly indicated in this publication. The reusable cup is filled with new liquid up to 3-4 times a day. The user rarely performs the process of cleaning and disinfecting lids/mouthpieces during such filling. Even if he does it, it is very cursory, and inaccurate because of the rush. Moreover reusable cups are exposed to a microbiological exposure in large clusters of people. After all, water-air bioaerosol has a very diverse microflora. A mouthpiece that has just been used can dry up to several minutes. At the same time, there can be several dozen of people in one room. An example of such a situation may be the use of cups with lids by students in lecture halls or employees in Open Spaces. There are biological and physical (temperature and humidity) conditions for the transmission of microorganisms. The composition of consumer’s saliva, roughness and type of material (from which the lid/mouthpiece is made), determine the strength of adhesion and the size and speed of creating multi-spieces biofilms.

The assumption was to check and compare the microbiological state of the lids/mouthpieces on disposable cups and repeatedly reused cups. For this purpose, the overall metabolic activity of microorganisms was assessed by measuring the amount of ATP using bioluminescence method, which is very fast, relatively cheap and easy to use in gastronomic conditions for microbiological detection.


Detection of microorganisms was carried from abiotic surface (PP/PS) on the lid of disposable and of reusable cups. It was a circular surface (r = 1.5 cm; 7.1 cm2) around the mouthpiece which is a contact point with consumer’s mouth. Types of examined cup lids: disposable, not used and not placed on the cup, so not touched by personnel (marking A, n = 37); disposable, not used but touched by personnel (B, n = 37); disposable once used (C, n = 37); repeatedly used after cold drinks and after room temperature drinks, e.g. water (D, n = 37); repeatedly used after hot drinks, e.g. coffee or tea (E, n = 37). Where “repeatedly used” meant adding the drink to the entire cup 3-4 times, drinking from it 1.0 to 1.4 L of drink, with at least 100 times contact of the surface around mouthpiece with the consumer’s mouth, with time of usage 9-10 hours. The collected cups were tested for up to 2 hours.

Additionally, swabs were taken from hands of personnel. The swabs were taken from randomly selected employees, from both hands (n = 25) during work, at 7 different gastronomical points. Swabs were taken alternately for traditional microbiological cultures and luminometric measurements. 

 1. The bioluminescence method

ATP bioluminescence was measured using a FireFly luminometer by Charm Sciences Inc. (Malden, USA). PocketSwab Plus swabs by Charm Science Inc. (Lawrence, USA) were used. The measurement procedure was consistent with the instructions of the device and swab manufacturers and described by Cais-Sokolińska and Pikul [1]. Bioluminescence was recorded directly at the site of smear collection.The total test time including the reading did not exceed 45 s. The results are given in arbitrary relative luminescence units (RLU) in 1 cm2.

 2. Traditional method – microbiological culture
The study of microbiological contamination of hand surfaces was carried out using the classical swab method. Hand swabs were collected by rubbing the inner surfaces of both hands, the areas between fingers and the outer parts of the fingers, including area around nails, with swab moistened with dilution fluid. The swabs were tested within 2 hours from the moment of collection. After taking swabs from employee, the tip of the swabs were placed in a sterile polyethylene bag containing 40 cm3 of rinsing fluid. The rinsing liquids constituting the initial suspension  were placed in the amount of 1 cm3 onto the substrates as deep plating culture, onto two parallel plates. The substrates was incubated in a WTB Binder microbial incubator (Tuttlingen, Germany). The number of microorganisms (cfu) on the surface of both hands was given as the multiplication of number of microorganisms in 1 cm3 of the initial suspension and the volume of the initial suspension (cm3).


On the hands of the workers putting on the lids, 2.93 ± 0.55 log cfu (CV = 18.76; P5-P95 from 2.71 to 3.16) was marked and 1684 ± 2149 RLU (CV = 127; P5-P95 from 797 to 2571) was measured.

The highest number of microorganisms that was marked on hands was 3.84 log cfu (4632 RLU was measured on hands). However, the largest amount of measured ATP is 8400 RLU (3.76 log cfu marked on these hands).

The analysis of bioluminescence measurements showed that the personnel hands did not cause a significant increase in ATP on disposable lids (P>0.05; Table 1). Further use of these lids increased the amount of ATP (2.5-fold; P>0.05). 36 to 6541 RLU/cm2 (mean 673.62 RLU/cm2) were measured on the lids used once. However, statistically there was no difference between these samples. There was a sharp increase in the amount of ATP on the lids due to repeated use. This was found irrespective of whether cold or warm drinks were drunk from the cups (P>0.05). The highest measured amount of ATP is 127 888 RLU/cm2. However, the large difference between the mean and median in this group and the large skewness ratio (2.32) indicate that this is not a normal pattern (Fig. 1). For verification, the Shapiro-Wilk test for distribution normality was used (W = 0,618). As a result of the categorization, it was shown that the largest group (77%) were lids with 134 to 25,686 RLU/cm2.

Table 1. The amount of ATP (RLU/cm2) on the lids of cups used once and repeatedly
Cup lid Mean P5 P95 SD CV
Control; A 76.16 a 55.63 96.69 61.57 80.84
Not used but touched by personnel; B 280.59 a 196.12 365.06 253.35 90.29
Used once; C 673.62 a 233.24 1114.01 1320.82 196.08
Repeatedly used after cold drinks; D 13 660.14 b 4325.73 22 994.54 27 996.21 204.95
Repeatedly used after hot drinks; E 18 105.49 b 9013.96 27 197.01 27 267.76 150.61

Different small letters in superscript in columns indicate statistically significant differences
at the level α = 0.05.

Fig. 1. Categorized histogram of the amount of ATP (RLU/cm2) on lids repeatedly used after hot and cold drinks


Up to 8400 RLU were measured on the hands employees' hands during work, which corresponded to 3.76 log cfu. Personnel’s hands were not the reason of increasing amount of ATP on the cup lids. There was a sharp increase in the amount of ATP on the lids due to repeated use. This was found regardless of whether they were cold or hot drinks.


  1. Cais-Sokolińska D., Pikul J., 2008. Evaluation of steel surface cleanliness level in dairies using the bioluminescence method. B. Vet. I. Pulawy, 52: 625–29.
  2. David D. Loschelder, Henrik Siepelmeyer, Daniel Fischer, Julian A. Rubel, 2019. Dynamic norms drive sustainable consumption: Norm-based nudging helps café customers to avoid disposable to-go-cups. Journal of Economic Psychology, 75: 102–146.
  3. Naik A., Lewis C. J., Allison K. P., 2019. Temperature dissociation of liquids in reusable thermoplastic containers - An eco-friendly scald risk? Burns, 45: 1621–1624.

Received: 2.06.2020
Reviewed: 26.06.2020
Accepted: 26.06.2020

Jakub Biegalski
Department of Dairy Products Quality, Faculty of Food Science and Nutrition, Poznań University of Life Sciences, Poland
ul. Wojska Polskiego 31
60-624 Poznań
email: jakub-biegalski@wp.pl

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.