Both should work fine, but I always recommend streaking onto agar plates first. If your stocks are contaminated this will often be visible on the plates but very hard to see in broth. Furthermore, if you are counting on doing growth assay and comparing growth to for example a mutant strain, or if you want to do some kind of gene expression analysis it is much easier to synchronize different strains when starting from single colonies.

good luck

Jesper

I don't know which is better but I have never had any problems with just streaking on agar plate. Why not test both?

It doesn't matter. In any case your stock is a clon so you will growth a clon. I usually grow them directly into LB by taking just a bit of the unfrozen stock with a yellow tip.

They will both work fine. Like Ruben mentioned you can take a small amount with a tip and add this to a broth size of your choice, or you can scrape some from the surface with an inoculating loop and streak this onto a plate. Most of the time you will probably be wanting to prep plasmid DNA from your clone or suspend cells for electroporation so directly using a broth will save you time. I hope that helps!

Both should work fine, but I always recommend streaking onto agar plates first. If your stocks are contaminated this will often be visible on the plates but very hard to see in broth. Furthermore, if you are counting on doing growth assay and comparing growth to for example a mutant strain, or if you want to do some kind of gene expression analysis it is much easier to synchronize different strains when starting from single colonies.

good luck

Jesper

As per my opinion and experience it is always preferred to go for streaking method on agar plate instead of directly inoculates them in broth. The rationale behind this is to avoid the enrichment of contamination if already in the glycerol stock. To revive pure culture you first streak the culture on agar plate than take well isolated single colony and inoculate in broth medium. Thus the revived culture will be pure.

I always take some frozen stock and use LB broth having antibiotics. People prefer to use streaking method but for me both worked in past..

Avinash... when the culture was in -80C they are already in stress condition and you are again adding antibiotic during reviving process. I think this could be ok for a clone. But What is your opinion if I want to revive a bacterial cell (lets say a competent call) which isn't antibiotic resistant?

I have always streaked them on suitable media, with or without antibiotic (depending on the strain). After strain revival prepare liquid culture for further experiments.

@Artur, I think you don't have any basic idea about microbiology. You wrote.."antibiotic should be present regardless of the method"....hold on a second my dear....then what about those cultures which are not antibiotic resistant ????

I also agree that there should be no antibiotics in the medium and the culture (glycerol stock) should not be though during streaking.

Hi, I suggest you to to go for streaking method on agar plate with appropriate antibiotic, pick a single colony and inoculate them in broth. this method helps you to avoid contamination.

God speed you

@ Artur Burzynski from Institute of Oceanography Polish......why suddenly u have deleted your comment??!! Where your great attitude have gone?? This is the problem with such an arrogant scientist like you....you should understand everyone is here to suggest..not to humiliate.

When I was doing this for maxi preps I would use a pipette tip to scrape a small amount of the glycerol stock and then innoculate a small amount of LB (usually 5mL) and then culture overnight then using around 1mL of that to innoculate 500mL of LB.

Ideally streaking on agar plates is best to revive cells. I always do this and after an overnight incubation, the cells always grows. Antibiotics should be added if the cells you are trying to revive contains plasmid with the antibiotic resistance. I have also seen people directly adding the glycerol stock to the culture broth, sometimes the cells grows but most of the time it does not. So I prefer streaking is better.

It is always good to keep bacteria in glycerol stock on dry ice. If you dont need to select them than scrap little amount of bacteria-glycerol stock with pipette tip and directly rinse in broath and let it grow. Advantage is this that you will save time. Sometimes direct in broath culture do not work (I dint know why?) than you need to streak on agar plate.

I agree with the comment D.P Nagar, streaking method on agar plate instead of directly inoculates them in broth.

I agree with most of hte comments here. First, streak your strain onto rich medium agar plate; then, select well isolated colonies and inoculate them in liquid medium for furhter experiments. The reason for that is to avoid the extension of a previous contamination. However, I have to said that, when you are in a hurry and don´t want to wait for the colonies to grow, inoculating the glycerol directly in liquid medium works well too.

Good luck.

If you are really sure that the bacteria are not contaminated. I prefer to inoculate the broth directly

Good luck

Most (all?) of your comments are addressed into a classic approach as recombinant cell growth and expression. In this case streaking on agar plates is the dominant.

Let me add for other users using wild type / industrial bioprocess strains that sometimes direct inoculation is the common way. I have experience with strains with really poor growth on solid supports. Then, direct inoculation (with precaution of contamination) is definitely a good choice

Look Debaprasad, let us believe that our technique to sterilize the broth or to practice aseptic technique is perfect and yet there are a good chance of contaminating the culture, because there will be very high mortality of the desired organism in the frozen culture. You should use the liquid broth to revive the culture but with a very short incubation time say of 2 to 3 hours only and then plate out the culture using extinction-dilution method on a suitable solid medium. It should work.

Recover at 37oC for one hour dilute and spread on a plate and pick a single colony for liquid broth is the belt and braces approach to prevent incurring extra work further down the line. If it's a glycerol that I am using on a regular basis then I tend to just innoculte LB with it, but for one that has been sat arounf for a while or is of unknown origin I tend to err on the side of caution to avoid extra work later on..

both are ok but i agree with previous comments that the best is to spread on a plate and pick a single colony just to be sure. Happy cloning! :-)

Dear Friends. Every one of you have justified your point for the query. I just want to add something here. I also prefer to first revive the glycerol stock on a solid medium. In addition, I like to streak the culture from glycerol stock on to a specific medium, say for example for an enzyme producer, the plates used for enzyme detection by agar diffusion method may be used. This will help in picking the healthy colony giving good enzyme activity for further work.

I wanted to add a comment to Jesper Nielsen's answer. Streaking out the culture and picking a single isolate helps to insure keeping the same line of the original culture going. The chances of choosing a colony that has mutated is less likely, and is especially important if you are growing the culture to add to the storage for liquid nitrogen. If transferring the culture after growth to the desired OD, you might do well to minimize transfer in case over multiple transfers a fast growing mutant happens to pop up. It may not be likely but if you work with the same strain or culture for years it is bound to happen at some point I would think.

i agree with most of the comments. For revive of the organisms from the preserved broth it should be streaking on any selective media, such as mac-conkey agar, CLED agar etc. so that we can easily differentiate the colonies and this will help for isolating the exact organisms.

I also agree with most of the comments. Revive the organisms on an agar plate before transferring a single isolated colony into your liquid culture. This will insure you do not contaminate your culture and I also found that inoculating directly into a liquid culture does not always result in growth. Having discussed this with a number of people, it can be that some bacteria do not grow well at very low densities and therefore starting with a freshly grown colony from a plate works better. However, as also mentioned above, if you are sure your stock is not contaminated and you are pressed for time inoculation directly into liquid culture can work.

I agree with most of the comments. We streak out a bit of the glycerol stock and then grow one colony in broth. Also just wanted to add to remember to keep the glycerol stock on dry ice while you acquire a bit for streaking/inoculating so that you don't thaw the glycerol stock and lose cells. Good luck.

If you find your stock is dead, you can still rescue the plasmid. Spin down the bacteria and extract the pellet as a miniprep. You can then transform competent bacteria.

I agree with the most comments. Streaking is better. remember to use Blood agar (or nutrients agar). Never use a selective media.

Keeping in view most of the comments above and my own experience, the better option is streaking on a nutrient rich agar plate. This will revive the organisms before transferring a single isolated colony into your liquid culture.

It is better if you start from streaking and obtaining pure colony to make a broth culture rather than directly inoculating them into a broth. You have to make sure the strain you are inoculate is not contaminated. Frequently used glycerol stocks may have some possibility of contamination. If you want to have a pure bacteria culture do not directly inoculate glycerol stock into a broth. Pure colony will give you a culture with good quality as well as rough idea of quantity.

Streaking first on nutrient agar is fine. Choose a single colony for further work. Never assume the whole stock is pure especially if you are in a lab with shared fridges.

Dear

besides, if you have old stocks or with low number of viable cells, you can use the broth culture medium to increase the number of live cells. After that put on solid culture medium to check contamination and obtain single colonies to use. Remember to prefer culture medium with high sugar to decrease the cells stress.

I agree with most of the comments. However, it really depends why you are reviving the stock.

If you are growing the bacteria in order to purify a plasmid, and if the glycerol stock originally came from a single colony, you can get away with reviving them in broth.

Start by adding a ~20 microliters of your Glycerol stock to 3 ml of a nutrient rich broth such as SOC and shake it at 37 celsius for ~8 hours. Then add the starter culture to 250 ml of LB with a selective antibiotic that your plasmid has resistance to. Shake the large culture at 37 celsius overnight and purify your plasmid the next day.

As long as you sequence your plasmid and do a test digest you should be fine.

(The probability of your stock getting contaminated with a bacteria containing the same plasmid resistance, the same restriction sites, and the same size is very low)

I agree with most of comments and I suggest both the two procedures to be used. You can simultanously, strike on agar plates to ensure purity and inoculate broth culture medium to increase the number of viable cells and should striking after and compare. Essential is to use enriched media to provide further optimal conditions for cultures

All the Best

Both techniques are valid. If your final goal is to get the DNA from those cells you may go for LB with the specific antibiotic to make sure you have nothing but your own cells.

the most reliable method is strike on an appropriate agar (LB, nutrient agar, blood agar etc.) you can be sure the purity of culture. then take a colony and inoculate it in broth media.

I always use this way as mentioned almost all comments.

Both methods are fine. If you need using the stocks to extract the plasmid,or make competent cells,or do shake flasks fermentation, re-streak on agar is better. But if you do fermentation tank, inoculate in broth is better.

Hope that's be helpful.

Your are interested in retriving the bacterial from deep frozen cultures (-80C).

Simply defrost and streak on agar plate. Observe your bacterial culture for any contamination. If contaminated purified before going for innoculation in broth. Culture in broth is generally done for a specific purpose. Good luck

No defrost; agar streak, one bead per plate, try to obtain isolated colonies and let them develop well before you start working on them. this allows you to examine each colony and enables you to tell whether the culture is still pure or not.

Also, be careful and examine the time-chart of the tank temperature: there may have been variations which may affect viability of some strains and let grow a contaminant!

Good luck!

One can do the both but it better to do first the streak on agar plate as it also help to avoid the contamination. As everyone agree with the streak one but both are fine as long as contamination is not an issues.

Dear All, 

All previous comment are OK according to me. My comment is similar to that of Monia El Bour: better both than only one of them. Nevertheless all depends on the bug, goal and conditions. Eg. when we melted a Chlorella vulgaris deep frozen cryo tube, half of it was put on agar plate - good for producing slant agars afterwards. The second half was inoculated to tryptone-yeast extract shaking flasks which excellently shows in one day if we have (nearly) any kind of "free rider".

I also agree with that streaking is better to check if you have any contaminants. But if you are sure your culture is pure, then use both at the same time-broth and solid medium. What is the bug you are working on??

In my case. I usually streak on agar first. After that I move to broth media that contain 20% glycerol. It seem to be good. They can survive more than 5 years. I recommend to make more than 3 stocks. Divide to working, stand by and original tube. 

Pha.

If you determine that your stock is non viable, do not despair.  Spin it down and do a plasmid miniprep of the pellet.  It is highly likely that you will recover the plasmid.  I did this for a tetracycline resistant stock many years ago (when I still had some hair) and even published it in Biotechniques.

I have done both ways. But I personally feel if it is essential to isolate aspecific clone. First let the cryostock grosw in LB broth, and once get good growth then, streak the bacteria in Agar plate with selection medium/ antibiotic. The colonies thus obtained will contatin the desired clone. Revival is also easy.

Unless I’ve missed it, is your agar of choice specific to the sample type or do you just go for non-nutrient?

According to my personal experience, streaking first the glycerol stock would be more reliable to get your transformed bacterial cell having GOI And Antibiotics resistance marker.

I failed to get protein when I went directly for 1 culture from glycerol stock.

Best

Ketul saharan

Debaprasad Parai

Better if you can go through a streak plate technique(with a earbud type cotton streaker for microbial plating) on a Nutrient Agar plate(commonly use) or over a synthetic solid media(if you are working with optimized media for growth of the specific organism). It will give you a clear cut idea if there is a contamination in the stored glycerol stock culture. Then if this preliminary examination is OK then you can go for culture on liquid medium like LB, NB or others and also can compare the growth pattern if you have already identified the organism.

I hope the information will help you.

Swaraj, please, could you better explain? thank you

@ Loredana Sigillo #

Thanks for the query, Can you be specific about your doubt ? I think the above explanation for the question is easy to understand ! In case you need more depth knowledge you need to be specific with your query.

Yes, sure... why do you write about a earbud type cotton streaker and not a common bacteriological streaker?

@ Loredana #

Your question is very logical, I appreciate your imagination.

I would like to share my way of innovating the science, what I believe is that glycerol stocks are stored at a very low temperature i.e. -20° or -80° C for a longer period of time. So when you will start the revival of the bacterial culture then there may be a chance that the bacteria will show slow growth for 1st & 2nd subcultures as it will take a little time for its adaptation after a cold shock(due to storage).

Hence you need to inoculate with a higher mass for its efficient viability and growth.

If you compare both the type of streaking tools for microbial culture as in the image you can conclude that the cotton earbud will hold a higher mass of cells compared to the normal streaker.

I hope the answer will enlighten your knowledge...

A little story referring to the cotton earbud / Q-tip:

In Germany the police tried to catch a criminal whose DNA was found at many crime scenes. He/She seemed to be very versatile in the kinds of crime he/she committed, including murder. After quite a long time they found out that the DNA detected was not left at the crime scene by the offender but belonged to an employee of the company producing the Q-tips used to collect the DNA... It was already there and had nothing to do with the crime.

What I want to tell with this story is: Be careful with non-sterile equipment, especially if using growth conditions without antibiotics!

How I used to revive a glycerol stock: Take the glycerol stock out of the freezer but do not let it thaw. Take a sterile (autoclaved) pipet tip or something similar to scratch some bacteria-containing ice from the stock and place it on the agar plate, where it will melt immediately. Repeat the procedure if you think you need more material. Spread it as normally. The non-thawed stock can be put back to the freezer.

Take about 100 micro L by sterile pipette and culture it on specific agar .

I am quite sure that the method of Shadman might be very little help.

See attached info may be useful for you!

Sorry to say: The story told by Vivicia is only partially correct and Vivicia draws the wrong conclusions. The equipment used by the police was STERILE, but it was not FREE OF DNA. This is why the police repeatedly found the same DNA in various crime scenes, which actually belonged to a female worker from the company producing the tips.

What you need for streaking bacteria or inoculation in liquid media is STERILE equipment. So Q-Tips are ok as long as you apply a safe method for making them sterile. Q-Tips may not be ok for transferring bacteria e.g. for performing colony PCRs of any other method that will amplify DNA.

Both are good approaches however, it really depends on the bacteria if it is a slow growth or fast one.

The agar approach allows you to check for possible contamination especially if your frozen stock or it is use quite often increasing the chances of likely contamination.

The broth approach will allow the slow bacteria to grow faster than it would on agar plates. They will have access to more nutrients and in particular if it is shaking

I agree with Stolz, that the equipments may be microbiologically sterile but not free from genetic contaminants which is the reason for confusion and wrong conviction that would follow.

Dear Debaprasad Parai

Both methods are fine, I prefer activating in broth then streaking on agar plate and selecting well isolated colonies followed by morphological and biochemical tests for confirmation

I have learnt a lot from the answers above. Thank you all.