Not sure if this would help you get at your research question. You can use phenolphthalein to observe ph change in the agar and use that as a metabolism indicator. Though metabolism doesn't necessary mean growth but it could be a useful metric. Very active bacterial colonies will metabolize faster, resulting in a color change from red-pink-orange-yellow, compared to slower growing colonies. You can then compair the zone of color change to the diameter of the colony over time.

A Method for the Detection of Changes in Gelatin Due to Bacteria

William C. Frazier

The Journal of Infectious Diseases , Vol. 39, No. 4 (Oct., 1926), pp. 302-309

Published by: Oxford University Press

Article Stable URL: http://www.jstor.org/stable/30083274

Hope this helps.

Colony count (called "CFU") is the most important parameter of measuring bacterial growth.

Dear Professor,

I want to measure the growth from agar medium.

I do not think the size of the colony is a good way to see the growth of bacteria! The size of the colony are 3 and is typical of most bacterial species.

I would suggest to recover the colony in a sterile saline solution of known volume and mix and then:

direct count under the microscope

CFU counts by plating again and discontinuation

approximated the number of bacteria with reading of the spectrometer

If you really want to measure bacterial growth directly on agar surface I think you can do it on agar slides at least for some bacterial species. There is a nice figure (figure 3.5) in the book General Microbiology (Allgemeine Mikrobiologie) written by Hans Schlegel. You can visualize the development of a microcolony of Arthrobacter pyridinolis on an agar slide and count the bacterial cells up to 22 hours of incubation. The figure was originally published in Kolenbrander, P.E., N. Lotong, J.C. Ensign. in 1976 Archives of Microbiology 110. page 239.

A good alternative to the colony size measurement is the rate of CO2 production: place inoculated agar plate inside air-tight container and periodically withdraw headspace air subsamples for CO2 analysis (GC, IR-analyzer, mass-spec or NaOH tittration -- whatever tool is available). Ideally would be to run continuous air through container and record CO2 production automatically by sensitive instrument...

Respiration activity is normally tightly related to growth (although not strictly proportional). You can also measure O2 consumption (Warburg respirometer) or heat production if you have microcalorimeter.

It is difficult to measure the bacterial growth in agar medium. The size of colony is a genetic characteristic of bacteria and it is not totally correlate with growth. If you need to know the growth in solid media you can quantify enzymatic activity for example by colorimetric assay or by hydrolyses of polymers such as starch, DNA etc. But it doesn’t seem easy.

I used to have issues with radial growth not showing a clear picture when dealing with some fungi. So I started off with culturing the strains on surface of broth in Petri plates, harvesting the surface mat by filtration on paper and measure the dry weight after oven drying.

You could probably do something similar with bacteria too if you have them as pure culture on plates. You can wash the cells off the plates with sterile water, pellet down the cells, dry them in oven and check for dry weight of biomass.

If its not about biomass, I reckon you can harvest the cells like I mentioned earlier. Then plate them out by dilution plating method to check the numbers of cells.

Not sure if this is of help but just my two penny worth.

Cool suggestions. I started to measure the biomass- wet and dry weight. Its working well and good. I ploted the growth curve vs weight and hours. i got nice curve. Thanks for your valuable suggestions..

Whoa there, first we should establish WHY you want to know the growth rate on agar. What bearing does it have on your research question? Agar-solidified medium is an artificial growth surface for microbes and any method of measuring growth rate is likely to be highly variable and, hence, inaccurate. So many factors affect microbial growth rates, on agar or not, so that's an added complication. What medium, what depth of agar, what initial pH, what temperature, what design of Petri dish (e.g. how many air vents), the list goes on and on!

1. May you can Raman Spectroscopy (RS), paper I attached here they used RS for the identification of microbes with agar plates grown bacteria. This also can work for quantification.

2. Pick the whole colony do the gram stains (the colony you want more information) and do the random counting (I never did this one, but if need more information in one colony this may work).

3. SEM tells you more detailed information one colony.

4. For stable colony (no change when used liquid fluorescent dye) you can use confocal microscopy with dipping lenses

We have measured fungal growth by liquifying the agar on the microwave oven and then filtering biomass through a milipore. I think this can be adapted to other kinds of surface cultures.

By doing triplicates it is possible to show the reproducibility of each measurement. But, for reproducible results you should spread the inocolum as lawn culture, in order to assure that most of the cells are synchronized and go through similar stages on the plate. This way, you can have measurable amounts of cells with similar cell density over the plate. The use of point inoculations produce very irregular results because the older central sections of the colony have different cell density with respect to the younger and peripheral sectors.

Check, Process Biochemistry Vol. 33, No. 2, pp. 103-107, 1998.

You should notice that the physiology of microorganisms grown on solid surfaces can be quite different to the physiology of submerged cultures. In particular, microaerophilic bacteria may not grow and some metabolic pathways could be induced in a particular culture medium. Also be aware of the problems of substrate diffusion in agar plates with different A/V ratios (different agar depths) since they can change the final thickness of the biofilm on top of the agar plate, affecting the overall physiology. The ancient model of uniform surface culture, proposed by Pirt does not seem to work too well in light of recent work from our lab. Thus, it is important to specify the culture conditions in order to have reproducible results.

Please check Wang et al. (2012) Angewandete Chemie (Int. Ed.) 51:1-5, 2012 (DOI: 10.1002/anie.201205715). The authors describe the use of a combination of nanosensors to follow and quantify bacterial growth on a Petri dish.

In Agreement with Frederic Gich, Helmut Brandl