Modified cold strong  ZN staining is good to my experience and commercial stains are available plenty in the market or can be prepared easily. Here among the staining and destaining and counter staining steps, destaining is crucial and sensitive one and one should take care while doing this step.  Time for destaining depends on the degree of intensity of the stain over the smear. Some times it may be less than 20 seconds.

Usually no problem with other two steps (staining and counter staining).

Finally you please check for the methanol fixation of faecal smear and make sure the sample should contain crypto oocysts.  

best of luck

 Hallo Muhammad,

you have to heat up the carbolfuchsin solution (over a bunsen burner)until it steames (5-10 Min), rinse in water, differenciate in acid alcohol (1% HCl in 70% EtOH) 1-3 min, rinse in water, counterstain in 0,1% Methylenblue 1 Min, rinse in water, differenciate in ascended alcohol, cleare in Xylene, mount in DePeX. Positiv controls are recommended.  Cabol fuchsin: basic fuchsin 1 g, EtOH 100% 10 ml, 5 % Phenol in water 100 ml (this is essential.) Good luck and best wishes, Ute

you can refer my article

how did you prepared carbol fuchsin without phenol.....????!!!! it is critical and important to make the stain to penetrate the wall of the oocyct.

 anyway kindly find and read the attachment file..... you will find two methods(hot and cold) . i prefer the cold method but please pay attention to the amount of phenol and basic fuchsin must to use to prepare the stain .

please don't hesitate to send any question, about cryptos i think i will be able to help.

Hi Muhammad,

I agree with both Ute and Morshed, the carbol fuchsine has to contain phenol (you should use "Ziehl-Neelsens carbol fuchsine solution"). I prefer the "hot" Ziehl-Neelsen, but know that for staining Cryptosporidium, you can also use the "cold" one.

If you use the "cold" one, you should leave the carbol fuchsine on the slide for at least 20 minutes, preferably even longer!

Kind regards,

Idzi

just 2 minutes in carbol fuchsin  are enough (as described for cold method in reference i attached above), i prefer the cold method because it's easier and give the the same results. (this from my own experience) 

i attached some photo of my work stained via cold method 

 Modified cold strong  ZN staining is good to my experience and commercial stains are available plenty in the market or can be prepared easily. Here among the staining and destaining and counter staining steps, destaining is crucial and sensitive one and one should take care while doing this step.  Time for destaining depends on the degree of intensity of the stain over the smear. Some times it may be less than 20 seconds.

Usually no problem with other two steps (staining and counter staining).

Finally you please check for the methanol fixation of faecal smear and make sure the sample should contain crypto oocysts.  

best of luck

Hi,

You may use modified ZN stain ( Cold one ) with high modification in phenol concentration based on my research.

Dear all,

Have a good day. Thank you for giving suggestions and comments. I really appreciate. I will try to follow the protocol given by Morshed. Thank you very much.

If you are staining faecal smears on glass slides and after staining the slide is clear after staining it sounds like maybe,as you suggest, it is possible that all the faecal smear was washed away in rinsing. We always used a round (disposable) lab stick...trying to apply the smear rolling the faecal matter on to the slide in a single pass. Then air dry them allowing enough time to be completely dry. ...We always used the modified acid fast procedure so I won't comment on the staining...but the stained material should be clearly visible on the slide after rinsing...gently...then air drying of course.

I completely agree with Dr. Venu Ravipati. I think if you had fixed the faecal smear with methanol before adding the Carbol Fuchsin, probably you had not seen the clear slide in the end. Again personally I prefer and recommend the cold method, as it is comparatively easier and gives good results. Most importantly, Carbol Fuchsin should compulsorily have phenol. Wish you all the best.

Hi all, I used water sample for the smear. The water sample which was prepared by filtration through membrane. Then, it was diluted with PBST and then continued with staining.

Hi there

We wanted to practice the ZN staining method and made Dummy smears with non MTB organism, now my question is if your smear is not made from sputum sample but a colony from culture medium, will you get blue background or not? As some slides stained blue while others did't...... was it because in the latter our smear got washed off?

If all the bacteria in the colony smear were acid fast , you will not see blue background. But this step of using M blue very important for knowing if other bacteria exist in the smear or not.

Thank you for replying but even in AFB positive slide, the background stains blue while AFB stands out as red or pink ..... isn't that so?

Sometimes the media that surrounding bacteria could to be blue.