During a recent relocation of Xylogenic’s laboratories, coupled with an interest in adding several new assays to our repertoire, we re-evaluated our procedures to verify that the banes of laboratory quality control—irreproducibility, sample instability, protocol irregularity and assay fragility—had not crept into our analyses. An old school alchemist would have referred to these demons as Death, Famine, War and Conquest. The greatest of these is irreproducibility, which must be managed by proper sample handling and rigorous adherence to clear, unambiguous and robust protocols.
In our industry, analyses of the fermentation process require constant monitoring, from analyzing corn grind and corn moisture prior to fermentation, to analyzing changes in sugar and carbohydrate levels and composition during fermentation and, finally, to determining sugar and ethanol levels at the end of fermentation. In many cases, the samples to be analyzed contain active yeast, active enzymes or both. Thus, proper handling of these samples is crucial if one is to avoid spurious results due to the unwanted actions of yeast or enzymes prior to analysis—not to mention potential activities due to contaminating microbes.
Typically, sample handling errors can be avoided by conducting the needed assays immediately after sample collection. Unfortunately, delays sometimes cannot be avoided—an equipment malfunction occurs, an unexpected emergency takes precedence, or you spend more time than anticipated impressing the office staff with your athletic prowess. If a second sample cannot be collected as a reasonable replacement for a sample left sitting, it should be noted clearly that the sample was handled differently than standard protocols, and data derived from this sample should be taken with a grain a salt.
If a delay between sample collection and analysis cannot be avoided, steps should be taken to ensure sample integrity. For our own analyses, we typically bring samples back to our laboratory in Indianapolis. With our transportation mode, samples can reliably be in transit for at least 24 hours without alterations to the samples. This can be accomplished by freezing a sample in a frost-free freezer. However, if the volume of a sample is large enough, simply placing a sample in the freezer may not allow the sample to freeze quickly enough to provide accurate measurements later on. To reliably bring any biological or enzymatic activity of a sample to a rapid halt requires rapid freezing of a sample no larger than necessary. A robust method to flash freeze samples is to place it in a dry-ice ethanol bath for several minutes and subsequently store the samples in a frost-free freezer. During one quality control meeting, we had a brief, but interesting discussion as to where we might find a source of ethanol for that dry-ice bath.
Once samples have been collected, they may be subjected to a variety of analyses. Each protocol will have its own requirements for sample preparation and sample abundance. In each case, the proper reagents must be prepared. Whenever possible, reagents should be prepared ahead of time and stored properly as specified, which may be frozen or at room temperature, concentrated or undiluted. Each reagent should be labeled with the precise chemical composition (or as accurately as possible), date of preparation, name of preparer and any storage requirements. Avoid abbreviations, unless following proper scientific or industrial shorthand. Protocols should be written clearly and in detail to avoid any possibility of interpretation. (Hmmm, it doesn’t say I can’t leave it on the bench at this point until Monday morning?)
Whenever possible, our protocols are written in “bite size pieces.” If possible, a set of steps is kept on a single page, ending at a point at which the sample can be safely stored or frozen and the protocol completed later.
Each protocol must not only be clear, but also clearly doable. For example, the required sample number should be limited to one that can be reasonably carried out—don’t set up 20 samples knowing that each sample will have to be subjected to a 10-minute analysis and you will only have an hour before the samples are ruined.
Make sure before you start that the necessary laboratory equipment is available and properly set up for use. Preparation will prevent mistakes down the road from having to hurry. Scientifically, it is okay to go as fast as you can; however, hurrying is going faster than you can, and should always be avoided.
Finally, whenever possible build the time constraints and expertise of your staff into each protocol. If everyone who may have to perform a protocol is comfortable knowing they possess the necessary skills and time to carry out an assay, the likelihood of errors can be minimized.
The goal of laboratory management and quality control is to ensure that every test that needs to be performed can be done in a rigorous manner by multiple staff members. By adhering to proper sample handling, developing clear and simple protocols and proper planning before every assay, this goal can be achieved.