I am not aware of your objective but for getting CFU I would suggest you :

1.If you are not getting any colonies in higher dilutions like you said in 5th , 7th and 9th than try plating by lower dilutions ..

2. if you are not getting any CFU in 1st dilution than after inoculation in 1ml TSB broth you can put in on shaker incubator for 2 hours or more time so that the bacterial load increase and then go for dilution and plating.

hopefully this will work ..

I am not aware of your objective but for getting CFU I would suggest you :

1.If you are not getting any colonies in higher dilutions like you said in 5th , 7th and 9th than try plating by lower dilutions ..

2. if you are not getting any CFU in 1st dilution than after inoculation in 1ml TSB broth you can put in on shaker incubator for 2 hours or more time so that the bacterial load increase and then go for dilution and plating.

hopefully this will work ..

Hello, several reasons can cause problems.

1. Dilution and homogenization as mentioned in the previous answer as well as

In decimal dilutions always take not less than 1ml from the previous dilution, well shake each tube before taking. Keep at 37"C for 48-hour maybe you will see any growth.

The problems also can be caused by

- Nutrient media and growing conditions.

- Any antibiotics or grow inhibitors in your sample.

Good luck

Karen A. Darbinyan Prakash Chandra Thank you for your response, sir. I will definitely look over the suggestions which you were recommended above. Karen A. Darbinyan Sir, I didn't add any kind of antibiotics or other factors in the media.

Wait till 48 hrs. If there is no single colony, there must be a reason that the microbial load in original sample is too low. Plating with lower dilution will work definitely.

Wait till 48 hrs. If there is no single colony, I think microbial load in your sample is too low. You can Keep sample inoculated TSB media in shaker at 37"C for 48-hour, If growth will come the media color will change.

You can do another thing, If you are not getting any colonies in higher dilutions plate, spread the lower dilutions plate. Another thing, please be sure that the spreader you are using should not be hot.

Md. Niuz Morshed Khan Thank you for your response. I will check it

Fecal sample normally contains very high numbers of bacteria. therefore even at higher dilutions colonies still develop. In my opinion, there could be following reasons for not obtaining growth at the said dilutions

1. pH of the media should be optimum for the growth of E.coli (near neutral)

2. the expiry date of the medium should be considered

3. Plate the nutrient media at bearable heat (if pour plate technique is followed)

4. The spreader (or L rod) should not be hot while spreading.

5. the individual could be under antibiotic treatment.

Lo primero que se tuvo que hacer, es una prueba presuntiva, es decir, incubar las 9 diluciones a temperatura de 35°c o 37° por 24 horas y observar los tubos positivos, es decir que tengan presencia de turbidez, CO2 (campañas de Durand) y presencia de metabolitos en el fondo del tubo (sedimento) y de estos tubos realizar las siembras en medios de cultivo apropiados.

I think maybe you can try the pour plate technique instead of the spread plate technique. Measure the medium temperature is appropriate before pouring. Another may be you can incubate the samples for a longer time and observe.