TSA (Tryptone Soya AGar) plates for environmental monitoring

L

LauraGchem

#1
Hi,

Am using Tryptone Soya AGar plates for environmental monitoring inside a clean room. These are supposed to be incubated for five days however this poses a problem on weekends.
Was wondering how others get around this?

Cheers,

Laura
 
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bio_subbu

Super Moderator
#2
Re: TSA plates for environmental monitoring

Hi,

Am using Tryptone Soya AGar plates for environmental monitoring inside a clean room. These are supposed to be incubated for five days however this poses a problem on weekends.
Was wondering how others get around this?

Cheers,

Laura

Hi Laura

Prior to weekend store the plates in refrigerator and again incubate the plates (after week end) in optimum temperature. During the refrigerated conditions bacteria will live but growth will not occur.

Regards
S. Subramaniam
 
M

mguilbert

#3
I agree with bio subbu about holding plates over the weekend stops growth. I was wondering if these are plain TSA plates or if they have had antibotics or other reagents added. If they are just regular TSA plates they should only need to be incubated @ 35 C for 18-24hrs according to one manufacturer. Or is the required by the standard that you are using.
 
J

Jimmy the Brit

#4
Re: TSA plates for environmental monitoring

Hi Laura

Prior to weekend store the plates in refrigerator and again incubate the plates (after week end) in optimum temperature. During the refrigerated conditions bacteria will live but growth will not occur.

Regards
S. Subramaniam
While this can be a good idea, make sure that you validate doing so, as the thermal shock of moving from 35 degrees to 2 degrees can kill some bacterial cells, leaving you no viable colony to subculture and identify. If your microflora is predominantly Bacillus species then you should be OK, however if they are Staphs and Micrococci then you may have a problem.

In my experience the instruction for 5 days is a minimum incubation time, so incubate for seven days inside a polythene bag to preserve humidity (bag will not become microaerophillic there is more than enough oxygen in plate head space). Perform an interim count at 48 to 72 hours, marking the interim count on the plate base, in case you get a swarmer which obscures underlying colonies. Other methods I have seen is an initial incubation period at 35 degrees for 48 hours followed by bacterial count, then a reincubation at 24 degrees (in a polythene bag) for a further five days to enumerate moulds, yeasts and other ambient growers.

Both work fine, but again you need to validate against your microflora and I would suggest a suitable test panel (USP/EP/JP panels are generally acceptable and I think they are now harmoniized which makes a pleasant change)

Hope this helps,

Jimmy
 

bio_subbu

Super Moderator
#5
I agree with bio subbu about holding plates over the weekend stops growth. I was wondering if these are plain TSA plates or if they have had antibotics or other reagents added. If they are just regular TSA plates they should only need to be incubated @ 35 C for 18-24hrs according to one manufacturer. Or is the required by the standard that you are using.
Hi

TSA (Tryptone Soya Agar) is the standard medium for microbial recovery in environmental monitoring program used to detect facultative, non-fastidious and aerobic bacteria as well as yeast and moulds. But the incubation temperature will vary for bacteria and fungus. Normally for detecting bacteria we should incubate 30 to 35 C and for fungus 20 to 25 C. But there is a possibility to get both the colonies of bacteria and fungus in same TSA plates. For avoiding this type of problems you can add antibiotics during media preparation and, after incubation you can find fungal colonies only. (if you want to detect only fungal colonies)

In environmental monitoring the incubation period may vary depend upon the standards and regulation. The duration of incubation generally recommended is 48 to 72 hours.

Regards
S. Subramaniam
 
Last edited:
M

mguilbert

#6
Hi

TSA (Tryptone Soya Agar) is the standard medium for microbial recovery in environmental monitoring program used to detect facultative, non-fastidious and aerobic bacteria as well as yeast and moulds. But the incubation temperature will vary for bacteria and fungus. Normally for detecting bacteria we should incubate 30 to 35 C and for fungus 20 to 25 C. But there is a possibility to get both the colonies of bacteria and fungus in same TSA plates. For avoiding this type of problems you can add antibiotics during media preparation and, after incubation you can find fungal colonies only. (if you want to detect only fungal colonies)

In environmental monitoring the incubation period may vary depend upon the standards and regulation. The duration of incubation generally recommended is 48 to 72 hours.

Regards
S. Subramaniam
Maybe I am confused where I work we use a Tryptic Soy Agar. However, the original post stated incubation for 5 days. In my experience plates tend to dry up if incubated that long. Sorry about any confusion I may have caused.

Regards,

Matt
 
L

LandA

#7
If you have a problem with the plates drying up you could look at using deep fill TSA plates, or position them in locations not subject to high air flow/change rates, if possible. They should be left for 5 days to give slow growers a chance. Putting them in the fridge could shock weak organisms so is not a good idea. We had people come in to check over weekend at one stage, but if you validate 6-7 days with a bag or deep fill agar you should be fine. Whatever you do decide, it needs to be validated anyway as the others have said.
 
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