Gram staining issues - (Mar/16/2016 )

We've cultured what we believe to be V. parahaemolyticus on TCBS agar and then subsequently on TSA. However upon gram staining we can never get the slide to stain pink. It always stains heavily blue/purple. Even with prolonged exposure to the decolorizer the stain remains. What could we possibly be doing wrong? We've also tried the KOH test and weve seen it doesnt work however studies have been done to show that this species rarely works with this particular test. 

And why do you believe it is V. parahaemolyticus when it stains Gram+?

And the KOH doesn't work in V. parahaemolyticus and does not work for your bug?

You should change your mind, there is no reason to keep thinking that you have V. parahaemolyticus

Because we have a grew it up on TCBS and even if it wasnt V. parahaemolyticus the agar should inhibit the growth of gram positive organisms. As I said. So at worst it should be stained pink but not necessarily be V parahaemolyticus

We had a clean sample that weve been using but we were trying to verify its purity

Did you stain directly from TCBS? Subculturing in TSA could have promoted the overgrown of small contaminants surviving in TCBS or direct contamination on TSA... did you reinoculate in TCBS to be sure?

Do you have any Gram controls for + and - to be sure about your stain?

Did you prepare the medium according to specifications? e.g. not autoclaving if it's a manufacturer mix (Difco indicates that)

Regardless of the Gram, there are few limitations listed in the Difco culture media manual:

 

1. On initial isolation, V. parahaemolyticus may be confused with Aeromonas hydrophila, Plesiomonas shigelloides and Pseudomonas species.8
2. Sucrose-fermenting Proteus species produce yellow colonies which may resemble those of Vibrio.9
3. TCBS is an unsatisfactory medium for oxidase testing of Vibrio spp.10
4. A few strains of V. cholerae may appear green or colorless on TCBS due to delayed sucrose fermentation.9

8. Bottone and Robin. 1978. J. Clin. Microbiol. 8:760.
9. MacFaddin. 1985. Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. Williams & Wilkins, Baltimore, Md.
10. Morris, Merson, Huq, Kibrya and Black. 1979. J. Clin. Microbiol. 9:79.