Streak plating is the standard method but it is worth looking at your cells using a microscope first - some bacteria stick together or even grow together in "packets" e.g. Micrococcus are usually in tetrads (4s). In these cases it may be necessary to use a method to brak them apart e.g. Passing them through a fine needle, mild sonication or adding tween. The key thing though is to not have too much microbial biomass on your plate otherwise colonies growing near each other can merge.

Once you have prepared your plate incubate it upside down for a day or two depeanding how quickly they grow then use a sterile loop to pick a single colony into suitable growth medium.

Hope this helps.

The only factor that will affect colony density is the number of viable cells you plate. Too many and you will get a lawn or merged colonies. There is no hard and fast rule regarding how to plate the cells except the source. If you are plating ligation products then as a rule I usually transform 5ul of ligation into 50ul home made competant cells, then add 500ul of LB and plate 250ul. If you are using cultured media as the bacteria source plate as little possible ( 1 or 2 ul diluted with media and plated will give you indiviual colonies)

Or simply plate a range of volumes after each transformation and take the appropriately spaced plate.

Use Petri`s plates with solid medium for bacteria, dilute suspension of bacteria in liquid medium and reseed the suspension on solid medium.

You will obtain isolates colonies of bacteria.

Using the streak-plate or pour plate methods.you apply a microbial culture( drop) to the surface of agar in petri plate and spread it with a loop or a bent needle(first technique).The second technique(pour plate)differs from streak plate in that the agar is inoculated when it is still liquid and colonies develope through agar.(not only on surface)

hi dear munirah,

go to this website; http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/Coliform_assays/Single_Colony_Isolation.htm

Hi friend,

u can use differential media meant for that particular bacteria of your interest and also use quadrant plate technique it helps you better.

Hi, try dilution plating.

Hi Munirah, the easiest ways to get single bacterial colonies are:

1. If you have the bacteria sample diluted on a media broth, just dilute this bacterial solution using the same media following 10 fold dilution down, and plating 100 microlitres per dilution on, for example, TSB agar plates and spreading it with a sterile spreader. Depending on the turbidity of the Bacterial sample, you will have to dilute it more or less. For a tipical overnight culture, you might have to dilute it at least 7 times, from neat to (-7).

2. Or you could do something much quicker if you don't need to count cfu of the sample, which is taking 10microlitres of the bacterial suspension and using a sterile loop sreak it on the top of a sterile agar plate, then take a new loop to do some more steaking just touching the previous one and going onto another sterile bit of the same plate, and repeat this twice more, by the last streaking (using always fresh loops) you will have isolate colonies perfectly defined after incubating the plate at 37C (depending on the microorganism) for 24h.

Hi Munirah,

I usually take 10 microliters of the bacterial suspension and using a sterile loop distribuite with a zig zag on top of agar plate. Repeat it three time more and by the last you will pick isolate colonies.

Good luck!

thanks a lot friends... I'll do my best

To get a single colony of bacteria, whether it is from a culture agar plate or culture broth, using a loop just touch a colony from a culture agar plate and then streak on prepared sterile agar, as you streak flame between each streak. For culture broth, just pick loop-ful and do likewise as above; you will automatically get an isolated colony.

The best thing I have found in my experience, to insure you have a single colony, is to not overincubate the plate. You should not incubate your plates longer than 18 hours, MAX. Incubating for longer results in overgrowth and merging of colonies.

To purify, take one of the smaller colonies (again, this insures that you have a colony from a single bacterium) and streak on a fresh plate. Again, do not overincubate! 16 hrs @ 37 degrees tends to be best, in my personal experience, E. coli, but check your plate periodically, as different organisms have varying generation times. They may require less time or more.

I hope this helps you. Good luck! :)

Try serial dilution and plating. There is a lot of literature regarding that technique in all microbiology books.

As many of our friends have already suggested please use serial dilution and plating sub-samples from the dilution. To purify your isolate use streak-plating and pick up a single colony at the end. Use the same streak plate method one more time to insure purity of the bacterium that makes the CFU. The incubation of your plate depends on the type of organisms you are dealing with. Good luck:-)

Hi Munirah,

for instance, if you start with an overnight culture of TSB or LB (18-20 h), prepare a 10-fold serial dilution until steps 7-8., and plate out 100 microl. from last dilution steps.

Purification using Streaking plate method on simple nutrient agar by incineration between each streak to separate and finalized into single colony.

All those answers are good, the one additional caution I would add is to be sure you know what type of medium your bacteria will grow on. TSB or LB is good for lots of microorganisms but not for all.

Hi Munirah

A good and easy method is to perform serial dilutions and spread with sterile glass beads (stirring gently) 50-100 uL in petri dishes containing 1Xagar +1X of the medium in which the bacteria grow.

Dear Munirah,

Just about all the answers you have gotten are fairly reasonable. I do have one question. What kind of bacteria are you dealing with? There are some species that may present challenges. Species that generate large amounts of exopolisacharides may be difficult since the "colony" may look like a large mucus deposit. The other difficulty is to have a bacteria that can't be grown using conventional methods. if these aren't your case then, the most reliable and simple approach is to do a serial dilution and plate the higher dilutions in favorable media (as in the media more suitable for your species). As mentioned before, pay close attention to the incubation since is easy to obtain confluence after a given amount of time, again depending on the species. Is important to try and catch them as early as possible to get the single colonies. only then you should have a single colony to pick. Good luck!

Hi Munirah! Are you isolating a single colony from a mixed culture or from the wild? If it is from a mixed culture you can easily do it by do serial dilution and plating the highest dilution depending on your estimated population of the original mixture. Just plate 100µ. if you are culturing from the wild use dilution of 10-3 and 10-4 for heterotrophs and 10-1 and 10-2 for functional groups using selective media.

To purify a single colony, i.e. a pure strain or clone, I would not start from a liquid culture. In a liquid culture you can never be sure that the clone or strain you want to isolate is not outcompeted during the incubation phase. Certainly when starting from mixed populations this is imortant (e.g. soil samples). Try to plate dilution series of your sample(s) so that you have plates with maximum 300 cfu on a 9 cm petridish. This is about the statistical limit not to have overlapping colonies invisible to the eye. From a good plate with nicely separated growth pick one colony with a loop and again plate it on a new agar plate. Many researchers would streak out in the shape of a pentagram, but other techniques will also do the trick (see biology handbooks). repeat this at least two more times.

To have single bacteria colony, one method is to streak bacteria around the petri plate, working in a circle and diluting the bacterial population as you go. The microbiological loop is either flamed, or a new loop is used.

You may use the "streak" method. Please see this link:

http://biology.about.com/od/biologylabhowtos/ht/streak-a-bacterial-culture.htm

Usually the streaking plate plate method, even without decimal dilutions is sufficient enough (decimal dilutions are quite useful for the inexperienced microbiologist but do add considerably to the work load). Of course it is essential to know which kind of bacteria you are going to work with since different techniques work better to different types of bacteria and if you are going to work with a certain type of bacteria then you should go for a selective medium for them. If you have a problem with mucoid colonies then a) do not overincubate, b) perhaps you can try some other types of agar forms like agar overlay or pour plate (especially in the last you certainly need decimal dilutions).

To isolate single colony inoculate pure culture in liquid broth in a peptone water tube incubate only for four hours and plate on a suitable medium. It works very wellj

You really don't know unless you use a micro manipulator microscope and resting cells to be rigorous about it.

Four way streak method is really usefull to get single colony.

quadrant method of streak plate is useful and you may even try dilution plating technique, which at times is very useful.

Before doing the streaking , make sure that the medium you are using is fresh and of the same composition.

Before doing the streaking , make sure that the medium you are using is fresh and of the same composition.

sometimes it is possible to obtain isolated colonies of bacteria by using seed agar plate, prepare hard agar (2.8 % agar-agar) layer first on petry plate allow it to solidify then take soft agar(1.5-1.8% agar-agar), cool it nearly 40-45oC. then inoculate soft agar with dilute culture (10-5-10-7) shake well and pour it on hard agar surface. Observe the plate after 24-48 hrs of incubation, you may get isolated colonies on agar surface, subculture each different colony on slant and characterise it. I hope that it will be useful to you in this regard.

Streak plating is the standard method but it is worth looking at your cells using a microscope first - some bacteria stick together or even grow together in "packets" e.g. Micrococcus are usually in tetrads (4s). In these cases it may be necessary to use a method to brak them apart e.g. Passing them through a fine needle, mild sonication or adding tween. The key thing though is to not have too much microbial biomass on your plate otherwise colonies growing near each other can merge.

Once you have prepared your plate incubate it upside down for a day or two depeanding how quickly they grow then use a sterile loop to pick a single colony into suitable growth medium.

Hope this helps.

mmy opinion.....there is no specific method......and my technique ....the one i use.......

plate QS.......slant..........and alternate min 3 rounds SC. and the type of loop mattters..... it shoudl be less than 0..2mm ID loop or less..... or better a pointed inoculation needle.

try it.....annd tell me it helps

Use serial dilution method

For isolate single colony inoculate pure culture in liquid broth in a peptone water tube

Please describe your problem in detail. This is actually a simple problem but without knowing the problem in detail, answers may not be what you require.

@Vaishali.....why such numbers? just curious to know...:-)

Its better to do serial dilution followed by streak plate method...if you are intended to isolate the specific character of bacteria, maybe you can try the specific medium for that bacteria only...such as Mc Conkey for gram -ve bacteria.

Simply do the streak plate method on the culture medium then incubate at the optimum temperature

Hi dear, download this video about Streaking Bacteria for a Single Colony form below site:

http://www.benchfly.com/video/126/streaking-bacteria-for-a-single-colony/

You must improve your handling by depending on a microbiologist in your university

handling is very important

There are different ways to get single colony. Just try to improve your handling to spread bacterial cells on solid medium on plate and after incubation and bacterial growth to pick up single colony. If you have specific question, let, please, know.

Thanks....It is known even to non-microbiologists and it applies to any particle distribution in liquid. I was curious for the specific number you used..nothing else...:-)

One easy way without serial dilution, is spotting the liquid culture on one side of the plate, streak out on just half the plate until entirely dry, and then go with the streaker just once over the other side of the plate. Even at high concentrations of bugs resulting in a lawn on one side can result in well isolated colonies on the other side of the plate. If you streak from glycerol, use one tip to streak one side of the plate, and use a fresh tip to streak perpendicular from the one side to the fresh side. Always results in well isolated colonies. Good luck!

This lab manual web text book covers the basics

http://www.bact.wisc.edu/outreach.php

You must improve your handling .

It can be done my streak plating, in sterilized environment (Laminar flow), using sterilized equipments and apparatus, you can streak your inoculum from a tube/flask culture contained in nutrient broth. Streak the inoculum on a piece of fresh nutrient agar (sterilized as well) using sterilized inoculum loop.

You can actually refer to any websites regarding on the methods on streaking because everyone's method are different. Then incubate your streaked agar in controlled temperature for a few days to weeks (depending on your species) then you shall see a single, isolated colony growing on the agar. And that will be your single bacteria species.

Try combination of making serial dilutions of the sample and plate streaking method as described in the video provided by the Moein Safari comment. Of course, everythig has to be done under sterile working conditions (using sterile lab equipment and laminar flow).

A suspension of microraganismo should be made. Then make a serial dilution. It is sown for all dilutions technique depletion loop on Petri dishes containing the specific culture medium for growth of the microorganism under study.

Streak plating is the best method, and between each streak try to expose the loop you are using for culturing to direct flame till redness then cool it and make another streak, this method help you to dilute or decrease the number of organisms between each steak

By streaking the bacteria on agar plate in 4 quadrant with flaming before each quadrant streaking.

I have question. Both my first degree and master degree were in Microbiology, unfortunately my practical skills about microbiology is not that great, i have worked six years in food industry and since then i went other discipline, now i want go back in Microbiology and i want to improve my practical can any body let me know where I have start or if you can give me suggestion in African hospitals where I can even work voluntarily. Please let me know if you know how or where I can do it practically.

Many thanks

Abdihakim, I have no knowledge of African hospitals, so I will speak quite generally.

I would recommend to contact directly the hospitals in your city, and the biomedical laboratories (which analyze some of the samples too). The problem is that every sample is very valuable, because it's not replaceable; so I don't know if they will take the time and money to train you and let you work with this kind of samples right away.

Maybe a short technical degree at school, like a technologist degree, will be a better place to start and refresh your knowledge and technical skills. Once you'll get your degree, you will probably have no problem to find a job in this field.

Good luck

thanks, Claire to your respond about the inquiry that i have made. I dont have problem to pay during time of my training, I gained my degree from UK. But currently I am in somaliland a break away region of north west somalia which has not yet get their recognition and there is no good standard hospitals one of the reason that i am going back to my field is the needs in here somaliland for proper diagnosis, please let me know if you know any hospital that i have these experience, and i really appraciated your respond, thanks let us get in touch.

Quadrant streaking with a loop flamed at each end...

Just you need to observe a microbiologist while streaking a plate and train your self to improve your handling and avoid contamination

Lets refrain from repeating what has already been told.....Or else we, who are following the post, keep coming back to the forum to see the same thing again and again....Thank you for your cooperation....Cheers...:-)

If you want to get nice single separate colonies then the following method will be quite helpful:

1. Make 10-fold serial dilutions.

2. Plate 100 ul onto the nutrient agar plate with few sterilized glass beads.

3. Shake plates back and forth to help spread the liquid all over the agar surface.

4. Take beads out and put the plates upside down in the incubator.

At least in one of the plates you will have very nice discrete colonies.

Enjoy!

Two perfectly good methods of obtaining single colonies have been given.

Note that the media you use will also affect the results as colonies on selective media may hide viable but less vigorously growing bacteria. So even if you start with a selective medium, sub-culture a single colony onto a non-selective medium to check for purity.

In plates where the colonies are not well separated the use of a sterilized needle to collect colonies that are observed as separate using a dissecting microscope or magnifying glass can be used.

Good lighting is essential when doing this work.

I am agreed with H. Davey.

Serial dilution or four flame streaking method can give the single colony

Streak plating is the best method to isolate single colony. If you are doing with a environmental sample, serial dilute the sample first and pick individual colony and streak it on the respective media.

For plasmid DNA that originated on a plate, we often recommend single colony isolation for DNA sequencing applications. Simply streak a plate using 4 quadrants as some have already suggested. Then isolate what appears to be a single colony and streak on a second plate. A single colony can result from multiple bacteria cells that stick together. Spreading a single colony on a second plate gives a better chance of isolating a colony produced from a single bacterial cell.

Dear for obtaining single colony u should go with serial dilution upto ten fold and do plating of each dilution & make care on handling ..u will defiantly get single colony in any of higher dilution plate

The best starting material is a relatively carefully see the colony on your primary plate. This colony should not show obvious signs of being multiple pinprick colonies that merged into a single CFU. Using a sterile loop, sample from the center of the colony and begin a heavy streak onto a new plate of appropriate agar media.

Single and pure colony can be obtained through dilution streaking for several batches. Pure single colony can be confirmed by looking at the colour, shape, eleviation and the plan of the colony.

Use a sterile incubation loop, touch the center of the colony and streak lines onto a new agar plate with your medium. Sometime, you may cleave the fragile agar using the loop in a wrong angular. You can consider to use a pipettor with a plastic tip and let the top of tip melt over the spirit lamp and form a small pellet. Cool down for a while and use the melted tip to streak lines.

Hope it helpful. Enjoy!

you can use serial dilution and streak from most dilute in your plate. Streak accurately to obtain single colony. It is better to sterilize your loop between each streak

Depending on the initial source of your inoculum, you may want to serially dilute (10-folds) the sample in suitable media (e.g. 1/4 Ringers) before plating the inoculum on suitable growth agar. Upon obtaining the first set of growth plates, you may (or may not) obtain plates with clear single colony inoculum types. If not, you can further isolate the individual colonies by carefully picking out the respective colony and streak a fresh plate using the streak plate method. You should be able to obtain individual colonies of the different colonial types.

You must used serial dilution method for getting single colony in plate culture.

you can make serial dilutions of your sample and then streak from the highest dilution. First streak the inoculum in one border of the plate. Then you should sterilize the loop and streak in a new direction, touching just a little bit the first streak you made. You should always try to maximize the streaking through all the surface of the plate. The re-sterilize the loop and streak again in other direction. That should work.

I posted this on Mar 26, 2013....I am repeating it again...Lets refrain from repeating what has already been told.....Or else we, who are following the post, keep coming back to the forum to see the same thing again and again....Thank you for your cooperation....Cheers...:-)

Lot of answers !!! God bless.... all paths leads to HIM.....Munirah you must have had referred some books or articles.....if not please do so, this will help in clearing your doubts and give you an insight about the principle behind the methodology. If you need more help regarding the write ups feel free.....Gud Luck!!

Streaking for isolation is often helpful that is, you take inoculum from a plate, streak across another fresh, sterile plate, flame your loop, allow cool, then streak from the end of this also across the plate at different orientation. This can be repeated 3/4 times. Alternatively, diluting your sample by a factor and plating out can yield single colony. Single colony is expected to be pure, this can be picked up by a sterile tooth pick.

Proper serial dilution upto 10^-5 and 10^-6 dilution .from 10^-5 take 100 microlitre and spread on the medium plate as per ur requirements with sterile spreader.Incubate it .After incubation with the help of a loop just touch at the centre and do four well streaking on a fresh plate .after incubation in the last zone u will get isolated single colonies

Try serial dilutions and always make sure that you let you plate dry before you invert and incubate

The best way has been diluting the culture as much as you can then plating. Do not give the cultures much time after plating otherwise the colonies will grow very close and merge. Streak plate individual colonies as they emerge in plates and do it well to also get individual colonies so that you can tell if the original colony was growing as a mixture of different bacterial

Just do serial dilution. Consult relevant literatures if you do not know how.

I agree with Dr Tajuddin.This is the routine method we follow to isolate the pure growth .Streaking for isolation is often helpful that is, you take inoculum from a plate, streak across another fresh, sterile plate, flame your loop, allow cool, then streak from the end of this also across the plate at different orientation. This can be repeated 3/4 times. you can refer Bailey and Scotts Diagnostic Microbiology

Try streaking if you have a liquid culture or dense colonies ( using a loop) or spreading ( using a spreader) if u have a liquid sample or drop plate if again u have a liquid sample. There is also pour plating. It all depends what is your sample. Where do u want to get a single colony from

Hazel Davey good answer .

Till now streak plate method is appropriate for geting single colony, in case your organism are producing exopolysaccharides it is difficult to get single colony.

Streaking properly, from the most dense to the least dense.

You mean single cell or single colony ! ?

Touch the sterile inoculation loop to mother colony and streak on fresh plate, incubate at appropriate temperature, you will surely get isolated single colonies. use the mixed streaking method.

serial dilution and plating is the best method. streak plate also gives good isolated colonies ,. this will depend on your initial inoculum also. you can change your loop for the second streak to dilute the number of bacteria. It also depends on which organism you want to isolate

Serial dilutions have worked wonderfully for us. We need to appreciate that most of the bacteria we work with, tend to clump together.

A single colony of bacterium in a culture is nest obtained by touching the colony among many by visualizing through watch maker's lens. This should be made as a practice among your staff and students. This never fails. You not only make an inoculation of this colony into a liquid medium but also make a tiny thick smears for staining purposes. Your further processes on this pure culture will result in excellent scientific results.

Dr. H. N. Madhavan. MD., PhD., FAMS., FIC Path.

There is mony methods ,for harvest of colony,depend on what the purpose of use for example,when we needed for vaxination