Thank you Anna.

I am looking for something that would stain both the extruded and intact spores.

I have not worked with Nosema, but have done a good bit of work with Enterocytozoon and Encephalitozoon.  Traditional staining is very difficult, and the technique used depends on whether you seek simple identification or structural analysis, quantitative assessment, etc.  For very specific identification, we use the multiplexed Immununofluorescent Antibody (IFA) and Flluorescent In-Situ Hybridization (FISH) techniques.  But, these are quite expensive and labor intensive, and yield identification precisely by no other information.  The attached papers give some details, but cite other publications as well.

Article Human waterborne parasites in zebra mussels (Dreissena polym...

Chapter Dreissenid Mussels

Hi,

I do not have personal experience working with these stains but I do know that there are a number of studies which have used stains on Nosema apis and ceranae in infected honeybees. Thomas C. Webster is the expert on Nosema staining in bees. I have seen a number of presentations from his students at conferences. I recommend contacting him personally if you still have questions about staining Nosema in bees. He has tried a variety of methods using different microscope filters to be able to differentiate live and dead mature spores in a bee gut samples that have been prepared from ground up dissected bee guts, one example can be found in the poster presentation attached.

To detect Nosema at all development stages I am assuming you would be working with the gut lining tissue which is the only place you will find the immature stages of Nosema. I do know of a colleague which has successfully used the staining method from the attached Higes 2007 paper to stain all stages of Nosema. This staining method is still considered the easiest and most effective way of staining both mature and immature stages of Nosema in tissue. Other effective staining methods for bee gut tissues can be found in the Fries 2013 paper which I consider to be a great resource to refer to in general when working with Nosema or honeybee experiments. Ingemar Fries is the expert to contact regarding the identification of Nosema spores and he is always willing to help, so I recommend contacting him if you need any help with the identification of the spore species. The staining methods in the poster presentation attached also looks like it may be possible to stain the internal structures of Nosema as well. I hope this helps you get started!

Best regards,

Chris

Hi

I think that the chromotrope 2R is suitable for microsporidia detection and diagnosis.

Regards

Hello Marie,

There are some methods, not yet mentioned which you could try:

Nitrate Silver Staining: http://www.ncbi.nlm.nih.gov/pubmed/19358851

Giemsa Staining: http://cmr.asm.org/content/12/2/243.short

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2863439&tool=pmcentrez&rendertype=abstract

Hematoxylin-Eosin Staining: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3356400&tool=pmcentrez&rendertype=abstract

Kind regards,

Richard

Nosema spores from in lepidopterous insects can be detected in their meconia with Bffalo Black staining (according to Frank M. Davis and Amanda Lawrence, Department of Entomology and Plant Pathology, Mississippi State University)

Technique for Preparing Buffalo Black Stain

 Step 1 – Measure out and mix together the following ingredients

0.15 grams of Napthol Blue Black

45 milliliters of methanol

15 milliliters of distilled water

30 milliliters of acetic acid)

Step 2 - Pour the mixture through a filter (Whatman’s filter paper or simply a coffee filter)

Step 3 - Store the mixture in refrigerator. 

Staining the Slide

The staining technique is as follows

 Step 1 - Place air-dried slides on warmer at 40◦ C

 Step 2 - Immediately flood slide(s) with Buffalo Black stain and let slide(s) stay on warmer for 5 minutes (do not let the stain dry on the slide) 

Step 3 - Rinse the slide(s) by gently swishing in a beaker of clean water, and

 Step 4 - Allow the slide(s) to air dry before adding immersion oil a cover slide and examination under the microscope.

Thank you everyone for your precious help!

One might see also a new request(2017-11), cf: https://www.researchgate.net/post/Is_there_a_histology_stain_for_staining_the_polar_tube_in_Nosema_apis_and_or_N_ceranae?

Given this study Article Comparison of Three Staining Methods for the Detection of In...

It shows that calcofluor white is the best staining method for microsporidia for it has high sensitivity in comparison with other examined methods hence can be used as primary screen test in diagnosis of intestinal microspridia.

Other than that calcofluor white's advantage is that it could be stored for long time in laboratory with little loss of fluorescence if solution stored in darkness. Another advantage of this staining method is, individual microsporidia can be distinguished in thick area of smear that may be overlooked by chromotrope or Gram chromotrope staining methods

There was a study conducted that compared different stains namely: Calcofluor white, Gram giemsa, Masson’s trichrome, acridine orange, Gomori’s methanamine silver, Gram’s chromotrope and modified acid fast chain in the four cases diagnosed with microsporidiosis on Hematoxylin and Eonisin and Periodic Acid Schiff’s stained sections of the corneal button.

Results showed that Gram’s chromotrope and GMS stains provided a reliable diagnosis of microsporidia. Therefore in terms of being cost-effective and the rapidness of the technique, the 1% acid fast stain and Gram’s chromotrope stain are the ideal stains for the detection of microsporidia.

https://bmcclinpathol.biomedcentral.com/articles/10.1186/1472-6890-6-6

According to BMC, In a study comparing special stains for the diagnosis of microsporidia, ie H & E, modified trichrome, Warthin Starry and Gram stain, modified trichrome was reported as the most effective [22]. In our series we were able to confirm the diagnosis using 1 % acid fast stain.

source:

Article Histopathological evaluation of ocular microsporidiosis by d...