How long do colonies on plates last

Being obsessive by training, we are trying to exceed measures of accuracy and precision in this exercise that the traditional methods cannot come close to matching.

The origin of those ranges is worth examination. Breed and Dotterrer published a seminal paper on this topic in 2. The matter of selecting plates to be used in computing a count becomes therefore a matter requiring considerable judgment.

The major paper from Tomasiewicz et al 3 provides an excellent review of the continued evolution of the appropriate number of CFU per plate from milk. They took data from colony counts of raw milk from three different experiments each dilution plated in triplicate and used to determine a mean-squared-error of the estimate for all plates.

It is interesting to note that although the authors note that CFU follow a Poisson distribution, no mention is made of any data transformation used to approximate a normal distribution prior to the use of normal statistical analytical tools.

Tomasiewicz et al provide excellent cautionary advice:. This range was established in the food industry for counting coliform bacteria in milk. The range is acceptable for compendial organisms, except for fungi. It is not optimal for counting all environmental monitoring isolates.

The recommended range for Aspergillus niger is between 8 to 80 cfu per plate. The use of membrane filtration to recover challenge organisms, or the use of environmental isolates as challenge organisms in the antimicrobial effectiveness testing, requires validation of the countable range. The upper limit of plate counts is dependent on a number of factors, as described previously. The major issues include the colony size and behavior swarming? The size particularly comes into play with plating a membrane for determination of CFU as the surface area of that membrane is so much smaller than that of a standard plate.

TNTC can be reported out several ways. It is not clear to the author how this is greatly superior to guessing. In my opinion this is an invalid plating and needs to be done correctly at a later date note I am strenuously avoiding the use of the word retest. This result invalidates the plating and therefore the test was not performed correctly. I know this is a hardship to the lab, who were trying to reduce the plating load initially by not plating out sufficient dilutions.

However, making a mistake initially is not a reasonable excuse to avoid doing it correctly after the mistake is recognized. There are methods available if you should want to accurately determine the upper limit for a unique plating surface or a unique colony type. This is based on the assumption that at the upper limit the observed numbers of CFU will fall off the expected numbers at some point see Figure 1. Figure 1. A central concern in this determination is the reporting of the Limit of Quantification which is what we are really interested in reporting against the Limit of Detection 1 CFU.

This is an important distinction if we are being held to specifications in the lower range. If countable colonies are present, but below the countable range, count them anyway and report an estimated count.

This is, in my opinion, the prudent course. The crux of the argument is that experiment studies have shown very poor accuracy in plate counts below 25 see above. This confusion between the Limit of Detection and the Limit of Quantification for plate counts has led to some very difficult situations as discussed below.

Ideally you would never see two separate dilutions with counts in the countable range, as the countable ranges cover a ten-fold range of CFU.

However, this is microbiology. Breed and Dotterrer 2 also used several dilutions if the numbers fit the QC requirements see below. While the argument can be made to use all counts, this is a stronger argument if triplicate plates are used and QC limits are in place to discard erroneous plates. A strong argument can also be made to take the dilution providing the larger number of CFU in the countable range.

This approach minimizes two concerns, that the errors in the estimates increase with increasing serial dilutions, and that the error in the estimate increases with decreasing plate counts. Use of the smaller dilution eg vs could be justified from this perspective. Periodically there are recommendations to establish Quality Control limits on replicate plate counts.

This method is not practical in the QC lab. Establishment of QC limits for plate counts works best if you have at least three replicate plates for each dilution. The average of the dilution replicates can be determined, variant counts hopefully no more than one plate per triplicate plating discarded and the final average determined. If you try this with duplicate plates you frequently end up with trying to average the results of one plate. Once the temperature is below the freezing point, however, cryoprotectants are essential to reduce cell damage caused by the freezing process.

The specific length of time that a culture will remain viable in a given storage condition is dependent upon the bacterial strain. Cell death during storage is inevitable but should be minimized as much as possible, which can sacrifice ease of use. Bacterial cultures that are used regularly i. If cultures will not be used for more than a few weeks, though, more long-term storage methods should be considered for maximum bacterial viability Table 1.

Culture dishes should be wrapped with laboratory sealing film plastic or paraffin and stored upside down agar side up to minimize contamination and to keep both the culture and agar properly hydrated.

Stab cultures are prepared by first sterilizing strain-compatible agar e. After the agar has solidified, a single colony is picked from an actively growing culture using a sterile, straight wire. As mentioned above, the temperature at which frozen bacteria are stored affects how long they can be stored while remaining viable. Freezing and thawing cells at an appropriate rate and maintaining the frozen stocks at the proper storage temperature help to minimize damage from the freezing process.

Also, the greater the cell density, the better the recovery is after thawing the cells. Cryoprotectants: As water in cells is converted to ice, solutes accumulate in the residual free water. This localized increase in salt concentration can denature biomolecules. Additives that are mixed with the bacterial suspension before freezing lower the freezing point and protect cells during freezing to minimize the detrimental effects of increased solute concentration and ice crystal formation.

Non-permeable additives used as cryopreservants, such as polysaccharides, proteins, and dextrans, adsorb to the surface of microorganisms and form a viscous layer that protects membranes, making these agents particularly useful for cryopreservation. Other commonly used additives include blood serum, ethylene glycol, methanol, skim milk, yeast extracts, and tripticase soy. Freezing samples: To prepare glycerol stocks, the glycerol is first autoclaved and allowed to cool.

The appropriate volume of glycerol is added to a suspension of log-phase bacteria and vortexed to dissociate the cells and ensure even mixing of the bacteria with the glycerol. When recovering strains with antibiotic selection markers, culturing them on selective media will ensure that the bacterial stocks were not contaminated. Freeze-drying: Bacteria can be freeze-dried by suspending log-phase cells in a lyophilization medium and then freeze- drying the suspension.

Not all bacteria can be successfully freeze-dried. The best way to determine if a strain is amenable to freeze-drying is to empirically evaluate its stability post—freeze-drying while maintaining a live culture as a backup. Simione, F. Key issues relating to the genetic stability and preservation of cells and cell banks.

J Parenter Sci Technol De Paoli, P. Biobanking in microbiology: From sample collection to epidemiology, diagnosis and research. Huba'lek, Z.