Bacillus Plate Count Variability
Microbial plate counts are conducted to get at best an approximation of the number of cells present in a sample. Since plate counting is a manual process there are several factors that can affect the results from technician to technician such as: quality of media prepared, incubation temperature and duration, the technician (technique and interpretation), homogenization, and plate count method employed.
Microbial nutrient needs vary by organism; however they are able to thrive in environments with minimal nutrients available. In the presence of a carbon source (ex: glucose), salt and water bacteria can maintain existence until it’s introduced to its optimal conditions. The microbe of interest in this blog would be that of the endospore Bacillus spp. This unique organism has the ability to lay dormant; resisting temperature changes, desiccation, and other unfavorable conditions for years if necessary until it is introduced to its optimal conditions to begin proliferation.
Spore-forming Bacillus thrives at temperatures ranging from 30°C to 37°C and colonies begin to grow in as little as 16 hours post incubation. In order to prepare a sample for enumeration a few steps must be taken to properly prepare the specimen due to its tendencies to agglutinate. A predetermined amount of the spray dried Bacillus powder will be dispensed into a buffered dilution solution. This suspension will be made homogeneous via shaking and homogenization for verified time duration that breaks cell clumping and allows for a more accurate enumeration of level of microbes in an ml/g.
Factors that will affect the sample recovery prior to plating would be diluents volume, accuracy of the samples weight, quality of media (media can be compromised with small levels of contaminates present; this will skew results obtained), and cleanliness of the work environment. Calibrations of pipettes are crucial, close attention should be paid to volumes dispensed by the respective pipettors used to plate the sample. Aseptic techniques must be employed by the technician as to not introduce any additional microorganism that will interfere with an accurate count. Accuracies and interpretation of colonies will vary by analyst; however a well trained plating technician will be able to reduce the occurrence of this with repetition and familiarity.
Technician variability is one of the largest factors affecting the counts. The work ethics of each technician could change counts up to the Millions, for example; are they properly mixing the batch and is it properly mixed for the right amount of time. As previously stated, if the batch is not mixed properly you will have some clumping or settling of bacteria. If the technician were to then sample you would undoubtedly get a higher CFU/g count if you pulled from the settling area than if you pulled from the top. Technicians may not be fully expelling all the pipettes liquid during the dilution step which could significantly affect counts. Simple counting errors such as mistaking two side by side colonies as one could effect the overall colony forming units. Some technicians become too relaxed or distracted and miscount colonies which ultimately affect the final cfu/g. In the biological industry, there is typically a 10-15% variance between technicians. This is why having two people plate a sample is necessary, so that a checks and balances system is established. Keeping up to date records on all activity and holding technicians accountable helps to reduce any error from the human element.
A plate count is a great way to assess the level of organism present in a sample. When working with a live specimen in a very manual process many of the factors mentioned above will affect the results; however adhering to methods in place and training will keep variations to a minimum; hence increasing reproducibility on results obtained.