Hi, it's better that you do not store your samples under the -20 freezer, if your sample is in liquid/semi-liquid state, and contains more than 60% of water. This is because at -20degC, your sample will be frozen and the water molecules will start to crystallize at around 0degC. Depending on the type of -20 freezer as well, some freezer has thermostat system which will repeatedly freeze and thaw your samples, and this will greatly affect the growth of microbes, as well as the structure of enzymes.

Crystallization of water molecules can also exert negative impact on the enzymes, whereby your enzymes will be denatured due to the formation of temporary bonds in crystallized water molecules. The enzymes might interact with the crystallized water molecules as well. This will cause further inactivation of enzymes.

Talking back about the freezer models, you must make sure that your -20 freezer is "frosted" type of freezer (and not "frost-free" type) as this type of freezers do not have the thermostat system as I mentioned above. Also, you need to make sure that your samples do not contain more than 60% of water content before storing them in the -20 freezer.

Hope all these information can help you. Good luck :)

So do you suggest that I rather take the measurements immediately? How about putting them in the refrigerator (4C)? Also, is it better to take samples from the same flask throughout the period or to have different flasks for each time interval. Thanks a lot.

The reason I am thinking of storing the samples at -20 or any other low temperature is to stop the action of the cells and preserve enzyme activity. I want to wait till the end of the experiment (after collecting all samples) before analyzing for OD, cfu and enzyme activity. That is why i asked if it is okay for me to keep the samples and analyze them all together at the end or to analyze each one immediately after collecting the sample (which would be more tasking because of timing). Thanks

In case that you have already done a validation of the bacterial growth curve, it might be an option to store broth samples at -20°C. However, I would not recommend to do so. As mentioned before the formation of water crystals could harm the samples and/or the freezing is taking to long.

Alternatively., it would be better to centrifuge the cells and to freeze them immediately after removal of each supernatant. In this case, I strictly recommend that you have already assessed the growth kinetics of the microorganisms by measuring dry and wet weight, absorbance at 650 nm, protein content, activity of enzymes of interest and the like. Since enzymes might be affected by freezing, metabolites, metal ions or else you also should know the stability of the enzymes of interest during freezing and storing.

Regards, Alec

Obviously, storing at -20 is not the best option. What about storing the samples (whole cells in broth) at 4C before doing cfu, OD and enzyme activity? Thanks all.

Hi Mushafau,

Yes, I am suggesting you to immediately do the sampling and measuring procedure immediately. I don't think you need to store your samples in the 4degC chiller either. If you want to store your samples in -20 freezer, it is better for you to extract cellulase first, followed by acetone precipitation. Because acetone has the ability to trap proteins (including your desired enzymes as well), and it can also be dried off after trapping, resulting a dry, whitish acetone powder that contains your cellulase.

This powder can be stored safely in -20 freezer because it does not contain water at all. Thus, causing no harm to your enzymes. You may need to search for protocols for this precipitation online if you came across this technique the first time.

May I know is cellulase extracellular or intracellular enzyme? And what is the microbe species you are dealing with? Thanks :)

Thanks Allan. The cellulase is extracellular and I am isolating it from a Gram-positive bacterium ( not yet identified).

Allan is right keeping the broth in -20 will definately have a negative effect on the growth and enzyme activity. It will also alter the CFU .Most bacteria prefer to grow at the temparature range 37-40 .Keep the broth in incubator at 37 and take OD at different time intervals at 660 nm using spectrophotometer. Simultaneously you can estimate the amount of cellulase produced care should be taken the enzyme culture filterate obtained after centrifugation which contain crude extract of enzyme should be kept in low temparature to avoid its denaturation .

From my experience, immediate analysis will give you reliable results. So consider doing the analysis immediately after taking samples.

OD and CFU should be done as soon as possible within your working day keeping your sample at 4C.

The sample ay (-) 20C can be used for enzyme activity only because at this lower temperature the ice crystals will damage the cells that changes OD and CFU.

The (-) 20 C can be used to measure OD and CFU only if the cells in spore form.