Hi there,

If you have the device to take pictures of agarose gel (black chamber+UVlight source) then it's pretty easy : you put your plate on the UV transilluminator and instead of switching UV on, you use a source of white light to illuminate the plate and then take pictures. The spots of yeast are made with 5 or 10µL of 10 times serial dilution from left to right starting with something like 107 cells per mL (cell suspension at OD around 1).

Thank you Dominique for answer.

you mean all these four dots are on same petriplate?

among these four dots . left one is 107 (ten raise to power 7) cells then 108 then 109 and right one is 1010??

please guide me. Thanks 

i have another small doubt ..........how to count yeast cells using OD600? if OD 600 is 1 then it is 107 (ten raise to power 7) ??

Hi again,

Well about OD600 and cell density, it dépends on your spec. It is roughly accepted that yeast culture at OD=1 is around 107 cells/mL ( the best is to calibrate your spec by reading culture OD and the performing cell numeration on agar plate and compare to find the exact Relationship between OD and cell density. Be careful the Relationship is valid for OD less than 0.5). So based on this assumption and if spotting 10µL of this cell culture then there should be around 105 cells in the spot as 10µL is 1/100th of 1 mL. So if you align 10µL spots of the 10 times serial dilution of this cell culture you should get from left to right (and provided all cells are viable on the agar plate) spots containing 105, 104, 103, 102 ,10 and 1 cells, OK?  So if you look at the picture first lane without salt pYES2/K601 (normally any cell spotted on this should grow), the far right spot contains 5 clones, OK? so the next on the left is containing 50, the third on the left 500 and the fourth one 5000, OK? So if everything is normal in the other conditions, there should be the same profile, right? If you have a look at different NaCl concentrations, this strain is perfectly viable (same clone density in spots) but if you look at the LiCl plates, one can see the cell viability is heavily compromised as the clone density and clone size are much less than in the control plate without salt. So in summary, you spot the same number of cells in different conditions and then compare the clone density and size after incubation. And then conclude!

Thank you So much Dominique Liger. I got my answer well.

I have another suggestion:

We always used a regular scanner. The dishes were placed without the lids onto the glass plate.  The orientation of the dishes is such that the agar faces upwards. 6 plate fit on a regular A4 scanner. Use software of your choice to cut and reassemble the slices as you need them.

When you handle a lot of strains it is best to dilute the strains in a 96well plate and use a 48-pin replicating tool (such as the one from SIGMA (R2383)) that will transfer a volume of some 2 micrometers per spot. The 48 pin tool will perfectly fit on a regular petri dish.

With a little experience (plate dryness is an issue) this really gives nice results. (see for example: http://www.jbc.org/content/283/12/7379.full.pdf+html

• wow sounds great idea. Thank you for suggestion J. Stolz . I will try this

Cannot you just place those petri-dishes on a nice/even black cloth (as background) and take pictures? Remove the covers of petri-dish to avoid reflections.

Of course, it should be possible to also take photographic pictures with a digital camera. Best results are obtained in this way:

use a black cloth or carton for background

mount the camera on a photographic stand for reproductions

use 4 lights for illumination to avoid reflections (see http://www.mot.be/en/opzoeken/smidstekens/tips)

take a picture of each plate individually

use manual settings on the camera to ensure that the exposure is identical

we always found it easier to use the scanner

@ J. Stolz,

1. I think black cloth is better than carton or paper, especially with those 'deep' black cloth. The texture of the cloth is also important for the quality of the pictures.

2. One thing is that the 'distance' from the object (petri-dishes) on the scanner glass plate to the 'Scan head' (mirrors, lens, filter and CCD array, see attachment) is fixed. So, when a scanner is used, the desirable sizes of 'colonies' on the agar cannot be chosen. While digital camera is used, the desirable sizes of the colonies can be chosen by adjusting the camera using zoom-in and zoom-out. Correct?

1. the pics in the JBC reference above were performed with a carton background.  I think they look ok. 

2. set the scanner to 600 dpi or higher and make a scan that shows the whole plate(s). load the image into some image software (photoshop, draw) and cut out the parts of the scan that you want to use with a rectangular tool. use the same software to rearrange the individual slices.

a slight problem with the scanner is that the colonies on the agar plates are outside the focal plane of the scanner (which is the upper side of the glass plate). however, we did never see much defocus in our scans. but this may also depend on the scanner model. 

Hi Subhash,

You've already got the answer. Once I did this kind of experiment.  When you have lot of transformants, prior to the plating load them to 96 well plate and dilute them in another micropalte with the same orientation (so you don't confuse). Then using multi-channel pipettor spot them on the plates with specific media you are interested.

If you don't have much facility to photograph, You can take the pictures by a digital camera. When you take photos of several plates try to take at the same heights. for this you can use burette stand. For the bottom you can use black cloth.

Good Luck!!!

1. Thanks, @ J. Stolz, for your response to my answers.

2. Personally, I think that to document a good result of the experiment should not be a problem. Instead, how to have an appreciable data is more important. And, the most skill-needed part in the experiment, I think, is to determine how much yeast cells you should use to spot (or dilution and spot) on the petri-dishes to demonstrate the function of your gene-of-interest. This may need some practice and work for a beginner.

to Juan-Yeu Yau:

as concerns the amount of cells to use:

we always use 4 spots per strain. the cells are diluted to OD 0.2 / 0.02 / 0.002 / 0.0002

we transfer these cell suspensions from a 96 well plate to the agar with the mentioned SIGMA metal replicator

in the final dilution, we always see 2 - 10 colonies growing

it is important to have a situation where single cells are spotted because only then it is possible to judge the growth speed of a strain on a plate from the size a colony has after 2-3 days of incubation

Thank you Akila Wijerathna Yapa for your answer.

@ Yuan-Yeu Yau and J. Stolz interesting discussion:-) thank you