Automated Bacterial Colony Counting for Clonogenic Assay

Introduction

Numerous dental diseases such as dental caries and periodontal diseases are closely related with the bacteria in our oral cavity. Taking dental caries as an example, dental caries is well-known a multi-factor disease, which occurred with both fermentable dietary carbohydrate and dental plaque bacteria. The Mutans Streptococci, one of the bacteria strains in our oral cavity, has been implicated as a major etiological agent of dental caries. Hence, it is critical to know what bacterial strains and the amount of them that are in the collected oral samples, such as the saliva and plaque samples. To identify and quantify the microbes in a sample, one of the most widely accepted function assay in both clinical and research laboratories, is the clonogenic assay (a.k.a. colony forming assay).

The clonogenic assay is achieved by pouring a liquefied sample containing microbes onto agar plates, incubating the survived microbes as the seeds for growing the number of microbes via binary fission to form colonies (colony forming unit, CFU) on the plates. The bacteria species can be distinguished by growing them on different selective medium, and the quantification for the amount of viable microbes in the sample can be measured by enumerating the number of colony on the plate, since each viable microbe can grow and become a colony. In this way, the identity and the quantity of the bacteria in a given sample can be determined. With this diagnostic tool, we can monitor the progress of the disease and even to indicate the susceptibility of future occurrence of the disease. Moreover, it also provides a basis to determine proper antibiotic agents used in medical treatment. Without a doubt, this assay is broadly used in biomedical examinations, food and drug safety test, environmental monitoring, and public health (Liu, Wang, Sendi, & Caulfield, 2004).

Though clonogenic assay is very useful, there exists a bottleneck that limits its throughput. The technical hurdle occurs at the final step, the colony enumeration step in the assay since it is a time consuming and labor intensive process. The reason is that there might exist hundreds or thousands of colonies in a Petri dish, but the counting process is manually performed by well-trained technicians. In Figure 1 (a) and (b), we show a typical 100mm Petri dish with hundreds of colonies grown in it.

Figure 1.

(a) Mutans Streptococci grown in a 100mm Petri dish with Mitis-Salivarius agar; (b) Escherichia Coli grown in a 100mm Petri dish with LB agar; (c) The hierarchical structure of objects in a bacterial colony image

In addition to the low throughput problem, the consistency is another issue. The manual counting is an error-prone process since the results tend to have more subjective interpretation and mostly rely on persistent practice, especially when a vast number of colonies appear on the plate (Chang, Hwang, Grinshpun, Macher, & Willeke, 1994). Thus, it is very important to have consistent criteria to avoid the fluctuations in results.

In order to increase the throughput and to provide consistent and accurate results, colony counting devices were invented in the market (Dahle, Kakar, Steen, & Kaalhus, 2004). We reviewed these counters available on the market (Advanced Instruments, ; Barloworld Scientific ; BioLogics ; ChemoMetec ; Colifast ; Neutec Group ; Oxford Optronix ; Perceptive Instruments ; Progen Scientific ; Synbiosis) and classified them into two categories.

The first kind of counter, the automatic digital counters (Barloworld Scientific), is widely used in most laboratories. However, they are not truly automatic since they require operators to identify each colony with a probe so that the sensor system can detect and register each count. Obviously, this kind of counter still heavily depends on the routine manual counting, and is of no use in solving the problems aforementioned.