# The microdrip method rapidly and efficiently enumerates bacterial colony-forming units in bovine milk

## TL;DR

A new microdrip method for counting bacteria in milk is faster, cheaper, and more reliable than traditional methods, making it useful for mastitis research.

## Contribution

The microdrip method offers a faster, cheaper, and more reproducible alternative to standard bacterial enumeration in bovine milk.

## Key findings

- The microdrip method reduces time and cost while maintaining reproducibility and precision.
- The method's sensitivity is comparable to quantitative PCR, with a detection limit of 10³ cfu/mL.
- Milk samples can be stored for up to 7 days at 4°C or −80°C without affecting cfu counts.

## Abstract

Summary: Investigating bacterially induced experimental mastitis requires reliable, precise, and reproducible enumeration of viable bacteria in milk. Standard microbiological methods to enumerate colony-forming units (cfu) are time consuming and expensive. In this work, we describe a microdrip method of cfu enumeration that not only reduces cost and time, but is also less variable, has increased reproducibility, and accommodates plating a greater number of technical replicates compared with standard bacterial enumeration methods. This suggests that the microdrip method is superior, considering statistical and practical considerations.

Summary: Investigating bacterially induced experimental mastitis requires reliable, precise, and reproducible enumeration of viable bacteria in milk. Standard microbiological methods to enumerate colony-forming units (cfu) are time consuming and expensive. In this work, we describe a microdrip method of cfu enumeration that not only reduces cost and time, but is also less variable, has increased reproducibility, and accommodates plating a greater number of technical replicates compared with standard bacterial enumeration methods. This suggests that the microdrip method is superior, considering statistical and practical considerations.

•The microdrip method reduces time and cost of cfu enumeration in mastitis studies.•This method has increased reproducibility and lower variability than the standard.•The microdrip method sensitivity is similar to that of quantitative polymerase chain reaction.•Milk samples can be stored for up to 7 days at 4°C or −80°C without changes in cfu.

The microdrip method reduces time and cost of cfu enumeration in mastitis studies.

This method has increased reproducibility and lower variability than the standard.

The microdrip method sensitivity is similar to that of quantitative polymerase chain reaction.

Milk samples can be stored for up to 7 days at 4°C or −80°C without changes in cfu.

In vivo microbial challenge models are an important tool to study the mechanistic details of bovine mastitis. A critical element of these studies is the enumeration of the bacterial load in milk to determine if interventions induce physiological changes that affect bacterial clearance. Herein, we use Escherichia coli P4 enumeration from milk in the context of mastitis microbial challenge models to show that use of a microdrip method reduces cost and time without negatively affecting rigor and reproducibility. We used E. coli P4–spiked milk samples as a model to test an alternative to Escherichia and costly standard plating methods. Importantly, linear regression analysis comparing the microdrip and standard plate count shows no difference between the methods or technicians, and Bland–Altman analysis shows enumeration via microdrip method has a slight positive bias, proportional across the tested concentrations compared with the standard method. Additionally, the microdrip method has a sensitivity of 103 cfu/mL compared with 104 cfu/mL for quantitative PCR. Economic analysis of consumable cost shows the microdrip method is nearly half as expensive compared with the standard plate method. Furthermore, these data also show milk samples can be stored at 4°C or −80°C without affecting colony-forming unit count, enabling batched sample processing. Taken together, the data presented here indicate that the microdilution and microdrip method can be used to decrease time and cost of E. coli P4 enumeration from milk without negatively affecting the rigor and reproducibility of the data. Future studies can develop this method for use in broader applications, such as validation of additional challenge pathogens for research purposes, clinical monitoring of mastitis cases, or bulk tank monitoring of pathogens.