I am looking for a protocol for total bacteria count, which include both viable and non-viable cells. The bacteria that I focus on are probiotic. Anyone have any advice?
As Liping Ii mentioned, flow cytometry is the best approach here. However, you will need to create non-viable and viable controls to make proper gates for the the flow cyotmetry software. There are limitations, because non-viable cells can be intact (intact membrane) or can be lysed. These two states will produce different absorbances in a flow cytometer.
Another option is to use Live/Dead Staining with fluorescence microscopy. Viable cells will be stained green and cells with membrane damage (presumably non-viable) will be stained red. The advantage to this is that you can differentiate between the viable and non-viable counts. http://www.lifetechnologies.com/order/catalog/product/L34856
If you have access to flow cytometry, you can use Live/Dead staining and program the software to count how many viable (green) and how many non-viable (red) cells there are in your sample.
Another (cheaper) method is to use a stain like Acridine Orange which will stain nucleotides in cells no matter what their viability status is. This will give you a total count of bacteria.
Anything with flow cytometry is most accurate, however microscopy is a cheaper alternative.
I hope this helps.
Flow cytometry is the accurate method, but is a costly method. Those who can not have flow cytometry equipment can done the total bacterial count by Haemocytometer. The protocol and the use is broadly uploaded on different websites. Just google search it.
If there is other cost effective option is there, I request the readers to provide.
If I am wrong some where please correct.
Thanks,
Is there any commercially available low-budget flow cytometer specifically designed for counting bacterial and yeast cells?
Dear Mr. Mesrop Ayrapetyan,
Do you think Acridine Orange can penetrate the peptidoglycan of gram-positive bacteria? Can I just stained the cells and count using hemacytometer under light microscope? What if the bacterial cells are being encapsulated? Do you think the dye is able to penetrate through the protective layer? Please advice.
Thank you.
Hi Shi Yiing Tang,
I came across this discussion while I was searching for a solution for my problem. I have exactly the same challenge as you do so i joined Researchgate so that I can get involved with discussions like this. I was wondering if you have gone ahead with staining your bacteria suspension with Acridine orange and if you were able to get a total count, I presume you would need a Hemocytometer or some sort of a counting chamber to calculate your counts. Please give me your views as i would like the opinion of someone that has experienced counting bacterial cell using a light microscope. Thanks in advance
I do agree that flow cytometry is a very reliable method for bacterial counting. However, microscopy is more commonly accepted as gold standard. Are there any good recent works comparing the two methods in accuracy?
Monfort (1992) already published a nice work on this topic with very good similarity between the two methods. Any more recent work?
Monfort, Beleux, 1992. Comparison of Flow Cytometry and Epifluorescence Microscopy for Counting Bacteria in Aquatic Ecosystems. Cytometry 13: 188-192.
can i use simple stain for bacteria with ordinary slide under light microscope?
Hi Alejandro Martin
I think the Bactobox from SBT Instruments is the right fit for you. The price tag in terms of instrument and sample analysis is favorable compared to Flow Cytometry. And it is super fast!
Sherlyn Tang, have you read the following article, I think it could be of relevance to you?
Why Flow Cytometry Could be the Probiotics Enumeration Method of Tomorrow - Microbiome Times Magazine
Enjoy reading.
Br
Thomas
Hello everyone, on top of this Q&A, I would like to know if Bactobox is able to discriminate between Gram+ and Gram- bacteria. In many fermentations, it would be crucial to understand if the desired Lactic acid bacteria (G+) are leading or taking the lead, discriminating them from other bacteria (e.g. acetic acid bacteria, G-). I see in the thesis of Dr. Christian Vinther Bertelsen that the modelling of impedance signals has been done accounting for shell distinctions between G+ and G-. Would it be possible to get only the signal of one of them?
Thanks a lot for the help
Marco
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