Dear Iftikar

Greetings

In Triple Sugar Iron (TSI) slant agar, the slant will turn yellow due to the increased level of acid production indicating carbohydrate fermentation. Hydrogen sulphide will also be produced due to the reaction of sulphur containing compounds. Hydrogen

sulphide reacts with the ferrous sulphate of the medium producing ferric sulphide giving a black precipitate to the upper layer of the slant. Hence, I suggest you, please check the media composition and ensure that all the ingredients including sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate are added appropriately.

Regards.

Feroze

Dear Iftikar

Greetings

In Triple Sugar Iron (TSI) slant agar, the slant will turn yellow due to the increased level of acid production indicating carbohydrate fermentation. Hydrogen sulphide will also be produced due to the reaction of sulphur containing compounds. Hydrogen

sulphide reacts with the ferrous sulphate of the medium producing ferric sulphide giving a black precipitate to the upper layer of the slant. Hence, I suggest you, please check the media composition and ensure that all the ingredients including sodium thiosulfate and ferrous sulfate or ferrous ammonium sulfate are added appropriately.

Regards.

Feroze

Thanks for you thoughtful reply. I am using TSI from himedia. All the ingredients mentioned by you are present in it.To check it again I have done a positive control with Salmonella Typhimurium . It has shown black colonies.

So, the other thing that you can control is if your strain produce the enzymes necessary for metabolism. I assume it is new isolate whom metabolism you are testing with these tests or is it established strain. In case of established strain I would suggest to consult the literature for its metabolic properties. If it is new strain there are always some mutants present in the nature. If this aspect of the bacteria is really important then perhaps may be do PCR for the presence of genes. 

Clostridium perfringens  has the ability to change its metabolic pathway depending on the final electron acceptor present under certain conditions and does not produce hydrogen sulfide. 

You want to make sure that it is incubated at the right temperature and atmospheric conditions.

#Benzecry

Madam, I am incubating slants at 37 c and pH is 7.4 under anaerobic condition.can you suggest any change.

try growing it at 44C --

check this article   http://www.sigmaaldrich.com/technical-documents/articles/analytix/clostridium-perfringens.html.

it could be very helpful

The answer is very simple : BECAUSE IT DOES NOT PRODUCE H2S.

The problem maybe that this strain is probably positive for H2S production but on TSI it is hindered in making it.  TSI has 10g/L of lactose and C.p. loves to ferment this sugar.  The acid production could be high enough to suppress the H2S production.  Why not use a medium made for C.p. such as Tryptose Sulfite Cycloserine Agar (FDA's BAM manual)?  Has no lactose and should give you good black colonies of C.P.  To easily see the black try to catch the colonies at the right time of incubation.  Test your strain by checking at 24, 36hrs, etc.  An example of this is some strains of Proteus that are H2S positive will not produce it in Lysine Iron Agar because enough acid is produced from the 1g/L of dextrose in the medium to suppress it.

Check your anaerobiose system. 

Deep inoculation with black colony growth indicates failure in anaerobiose.

TSI supplemented  with egg yolk agar is very good to identify C perfringens (black colony with lecithinase activity)

The C.p. medium, tryptose sulfite cycloserine is great for this bug and has egg yolk in it for lecithinase activity as well as H2S (black colonies) activity. Has D-cycloserine as a selective agent to help keeping your C.p. pure.  Great selective/differential plate medium for C.p.  Of course (see previous ans.), no lactose.

Thanks everyone.but the question still persist why c.perfringens is not showing black colonies on TSI?I am already doing TSC where it is positive. I am still not able understand why H2S is not produced.

1) As I understand anoxic (or limited amount of oxygen) conditions are required for formation FeS. I would recommend to try to use not slant shape of agar but straight in the tube. Inoculate melted agar at ~ 40C and wait. If sulfide is produced then you might observe FeS in the deeper parts of the straight agar.

2) very low pH should be avoided. From one side Fe2+ is chemically stable at low pH even in the presence of oxygen, from another - FeS could be dissolved!

@ Alexander sir,

The whole system is in a anaerobic jar. Therefor the first point has been taken care of.It might be a issue with the pH as I have not encountered yellow slant and black colonies together in TSI. For eg. Salmonella serotypes.

Based on what you have said, you are getting H2S production on TSC agar from your C.p.  You want to know why this same bug does not produce H2S on TSI.  On TSI, it must be the low pH due to lactose fermentation that is inhibiting H2S production.  This seems to be the case, thus nothing wrong with your bug. Just use TSC.  Why continue with TSI where you are going to get low pH?  Avoid using TSI.  Just as Dr. Galushko said above, "very low pH should be avoided".

@ Robert

Sir,I am avoiding TSI and confirmed the production of H2S by doing lead acetate test. I am doing these tests for confirming biochemical properties. For isolation I am doing Blood Agar.