Sunday, September 30, 2007

Lab Techniques

Hi everyone,

Like Jiaxin and Johanna, I am also the ‘supervisor’ of the DRP students. Shahirah and me are taking the group that will be doing a project that involves proteases secreted by Stenotrophomonas maltophilia at 37oC. We would have to teach them how to prepare the necessary media, teach them the things that they do not know (eg. how to streak in the proper way) and guide them through the experiment so that it is successful.

This is a very simple project and the purpose is to find out which are the strains that produce a lot of proteases. Strains that produce a lot of proteases might be more virulent as the proteases might be involved in the degradation of the host proteins involved in the immunity eg. Immunoglobulins (Ig).

Principle:

After growing the colonies, the colonies will be transferred onto milk agar. If the strain produces proteases, the proteases will digest the milk proteins and a clearing that looks like a halo around the colony will be observed.

Procedure:

Day 1 - Streaking of environmental strains

  1. Streak the strains onto LB agar (Plate is divided into 2 as there are only 2 strains and only abit of the colony is needed)

  2. Incubate the plate at 37oC for 48 hours (As environmental strains do not grow very well at 37oC, more time is required for them to grow)

Day 2 - Streaking of clinical strains, negative and positive control

  1. Streak Escherichia coli (negative) and Pseudomonas fluorescens (positive) onto a LB agar each

  2. Streak the strains onto LB agar (Plate is divided into 4)

  3. Incubate at 37oC for 24 hours

Day 3 - Transferring of colonies to milk agar

  1. Divide and label the milk agar appropriately


  2. Use a sterilised toothpick to touch the colony and then dab on the milk agar gently

  3. Incubate the plates at 37oC

  4. Observe for halos (This will take about 3 days of incubation)


That’s all! Thanks=]

Chua Ming Boon

Tg01

0503197F


9 comments:

royal physicians said...

hey girl....wanna ask u apart from milk agar..is there any other agar that can be used??

njoy ur last wks of SIP:)

nur zahirah
tg02

Distinction in Disaster! said...

hi ming boon

wah so young become supervisior le ah, hope the student don't suffer in your hand...

i would like to ask why Escherichia coli (negative) and Pseudomonas fluorescens (positive) are used as the control instead of other type of bacteria?

Lizzie

royal physicians said...

hello!

I have the same qn as Zahirah actually..is there any other kinds of agar for this experiment?

Thanks!

kangting 0503331A
TG01

VASTYJ said...

Hi Ming Boon

Since QC is done with the positive and negative controls, is there any blank control done for the LB agar?

Ying Ying

J.A.M.M.Y.S said...

Hi Zahirah and Kang Ting,

Milk agar is a general agar used. Yes, there are other agars such as butter-fat, cream and spirit blue agars but these agars are more specifically for the detection of lipases.

Hi Lizzie,

The reasons that we choose these 2 bacteria is that we do not have a huge variety of bacteria and these 2 are very easy to culture. Actually E. coli also secretes proteases but this E. coli strain bought from ATCC does not secrete and therefore is used as a negative control.

Hi Ying Ying,

There isn't any blank control done. However, after preparing the agar, we will incubate some of the plates as control.

Hope I had answered your questions=)

Star team said...

Hey,

I'd like to know whether the environmental strains are infectious to human? Besides immunoglobulins, are there other proteins that these proteases target in the host? Thanks.

Yong Yang
TG02

ALsubs said...

hi

so if the bacteria produces more protease, there will be more halos? can this be a form of quantitation?

cass TG02

The Lab Freaks said...

hi girl

since u only use a toothpick to trasfer the cells to the milk agar (which is sooo small), will there be enough proteases secreted in order for the halo to be clearly observed??

Suat Fang
TG01

J.A.M.M.Y.S said...

Yong Yang,

The environmental strains are not infectious as they can barely survive at 37 degree celsius. The proteins targeted will depend on the type of proteases secreted.

Cassandrea,

There will only be 1 halo. Its actually a clearing surrounding the colony. Yes it can be a form of quantitation but it is by measuring the diameter of the clearing. The more proteases secreted, the bigger the diameter.

Hi Xue Fang,

Yes the starting cell is very small. For those strains that secrete a high amount of proteases, the halo can be clearly observed but for those strains that do not, yup it cannot be clearly seen. That is why we are going to use an inoculating loop and streaked about 0.3mm onto the milk agar.

Ming Boon