Get starter culture

MPD-5

There are several ways to get a starter culture for your bioprocess. We use a specific input material and a defined workflow, to transform initially dormant cells into active cells with enhanced replication capabilities.

In the table, we summarize the pros and cons of different types of input materials. Details are given in the sections below. You can also jump straight to the step by step workflows in the subpages:

• From cryo stock

• From agar plate colony

• From liquid culture

• From CRM product

Most workflows result in starter cultures after overnight incubation, but by changing the seeding concentration you will often be able to get a same-day starter cultures.

	Pros	Cons
Cryo stock	Locks down genetic consistency Enables easy standardization Simple protocol with few steps	Potential risk of contamination Concentration of culturable cells may change during storage and re-stocking Requires a -80 °C freezer
Agar plate colony	Simple, low-cost Low risk of contamination Possible to add selective growth conditions and pick colonies with desired phenotype Does not require -80 °C freezer	Adds an additional incubation step that delays the workflow Challenging to get accurate counts because bacteria may be partially encased in biofilm and or agar.
Liquid culture	Fast and convenient Accurate enumeration of total cell concentration Does not require -80 °C freezer	Risk of contamination Risk of mutations occurring over time if subcultivation is repeated extensively May contain mixed populations
CRM products	Convenient, ready-to-use formats for consistency and traceability Often comes with well-defined bacterial concentration	Higher cost Limited shelf life May be difficult to distinguish between live and dead cells.

Cryo stock

Cryo stocks are one of the cornerstones for good manufacturing practices (GMP) in biotechnology mainly because you can effectively "reset" a culture to a consistent starting point each time. This ensures low passage numbers and thereby minimizes the risk of genetic drift. Cryo stocks are convenient, as a large bank of individual vials can be stored, with a vial being used each time a culture is required.

Using cryo stocks as input also comes with some drawbacks: The freezers are relatively expensive and culturability may drift over time which leads to inconsistent starter cultures. When the stock is depleted, re-stocking is necessary, which may lead to differences in growth dynamics. Finally, some of the individual cryo stocks may be contaminated - there is little change of spotting this contamination early because the cryo stock is added directly to the starter culture, .

Agar plate colony

The streak plate method is a straightforward, cost-effective, and reliable technique for obtaining discrete colonies and verifying the purity of bacterial stocks. The primary advantage of using a colony as input material is the reduced risk of contamination.

A downside to the agar plate step is the potential for significant delays in the protocol, as bacteria need incubation time to form colonies. Additionally, accurate determination of bacterial concentration can be challenging since the cells may be partially encased in agar or biofilm-like clumps. Extensive disaggregation is needed.

Liquid culture

The fastest way to get a starter culture is to do a subcultivation from an existing liquid culture in exponential or early-stationary growth stage. This substantially reduces the lag phase duration and may be relevant if your goal is to get an early-exponential starter culture.

It can be risky to use a liquid culture as input material due to its inherent lack of uniformity: Within the culture, both healthy and stressed cells might coexist, and genetic variability can lead to differing fitness properties. Over several passages, these differences might cause genotype drifts, resulting in inconsistent outcomes. Another drawback is that contamination may be challenging to detect: Unlike the streak plate method, real-time detection of trace contaminants in a liquid culture is challenging.

CRM product

Ready-to-use certified reference materials (CRMs) are available from different providers like Microbiologics, ATCC, bioMérieux, Merck, Zeptometrix and Thermo Scientific.

In principle the pros and cons of the CRM products are similar to the cryo stock option, but they offer convenience and reliability with the ready-to-use, certified formats. This typically comes at a relatively high cost, especially if a large supply is needed.